Polypeptides useful for appetite suppression

ABSTRACT

The present invention relates to polypeptides obtained from bear derivative isolate which are useful in suppressing appetite. The polypeptides of the present invention are most preferably between 12 and 13 amino acid residues in length and a mass of about 1249. The present invention also relates to a method of treating obesity by administering to obese subjects an effective amount of the polypeptides of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of application U.S. Ser. No. 10/841,710, filed May 7, 2004 now U.S. Pat. No. 7,060,300, which application is a continuation application of U.S. Ser. No. 10/232,880, filed Aug. 30, 2002 now U.S. Pat. No. 6,855,337; which is a continuation of U.S. Ser. No. 08/833,096, filed Apr. 4, 1997 now abandoned; which is a continuation-in-part of U.S. Ser. No. 08/470,750, filed Jun. 6, 1995 now abandoned; which is a continuation-in-part of U.S. Ser. No. 08/259,788, filed Jun. 14, 1994 now abandoned; which is a continuation-in-part of U.S. Ser. No. 08/079,089, filed Jun. 17, 1993 now abandoned, wherein said applications are incorporated herein.

FIELD OF THE INVENTION

The present invention relates to polypeptides obtained from bear derivative isolate which are useful in suppressing appetite. The present invention also relates to a method of treating obesity by administering to obese subjects an effective amount of the polypeptides of the present invention.

BACKGROUND OF THE INVENTION

It is known that denning, fasting black bears, fasting polar bears, and pregnant female polar bears who den possess blood factors that can recycle harmful body waste products back into usable protein for building tissue, and that denning, fasting black bears can continue to build bone when the bear is immobile for months at a time. Upon isolating the substance which controls this phenomena in the bear, there is the possibility that the same can be used to prevent toxic buildups that endanger humans with kidney failure that now require the stressful, expensive treatments of dialysis and kidney transplant to sustain life. The isolate (BDI) also includes the possibility that it can prevent protein breakdown which leads to life threatening situations in humans suffering burns and trauma.

It is believed that such knowledge can lead to strategies to combat bone loss, which afflicts millions of middle aged and elderly people, especially post-menopausal women and astronauts in weightlessness of space. Loss of bone mass in space is one of the major problems that prevents long term space flights by humans.

Bears preparing to enter the denning phase go through a period of hyperphagia during which they eat enough food to store enough fat to last through the denning period. During denning, bears do not eat, do not drink, and do not urinate or defecate. Exiting the den after a four to five month period, the bears resume normal eating patterns. Knowledge and/or the isolate (BDI) may be useful in developing strategies and/or products for the treatment of eating disorders such as anorexia nervosa and bulimia.

Black bears in particular, during their three to five month denning, show a reduction in body temperature of at least 1.13° C., remain alert and expend energy normally; yet they do not eat, drink, urinate, or defecate and exhibit no problems with waste building to toxic levels. Other mammals, including humans, can recycle some waste, but under similar conditions must quickly rid themselves of the rest of their waste or die.

It has been determined that bears in a non-denning state during summer months are induced to produce the isolate (BDI) after 20 days of fasting, even though they are allowed to drink water. Under these circumstances, bears urinated and did not exhibit the tranquility associated with a denning bear.

Other mammals (including deep hibernators such as ground squirrels who continually awaken throughout hibernation and generate waste they must get rid of) break down protein mainly from muscle to supply energy and other essential nutrients for life. This process not only depletes body muscle, it also releases the toxic form of nitrogen as ammonia. Mammals, including humans, convert the ammonia to urea, which is much less toxic but must be eliminated in urine. During denning, black bears also produce urea, but close this loop and recycle the urea nitrogen back into protein. They produce no waste and maintain muscle mass while eliminating the need to urinate or defecate. The process is so efficient that normal urea concentration in blood decreases and body protein increases. The bear is the only animal known that fasts completely (no food or water) yet ends a 100 day or longer fast with a little more protein (lean tissue) than when it started. During the denning period, the bear steadily consumes body fat that had been stored during the pre-denning period.

This unique response extends to maintenance of bone mass. The bear shows no bone loss even when supine over more than 100 days. In contrast, deep hibernators lose bone and exhibit osteoporosis when hibernating. The bear does not develop osteoporosis and is able to maintain skeletal integrity despite the harsh conditions. Under similar stimuli, humans would suffer severe bone loss.

Taken in the context of the foregoing, it is a desirable forward goal in the treatment of human ailments to be able to isolate the bear derived isolate (BDI) which permits the foregoing phenomena in bears, and to translate it into meaningful metabolic and curative processes in the human.

These goals appear possible. For instance, a bile salt produced by the bear has been shown to improve liver function in humans with the fatal disease of primary biliary cirrhosis. In humans, this bile salt also reverses serious rejection reactions against bone marrow transplants. Further, this bile salt, ursodeoxycholic acid, is the most effective dissolver of human gall stones. Thus, a isolate produced by bears has direct positive application to human disorders.

Experiments and observations directed to studies in denning bears have been under way for more than 23 years. During that time, it has been established that the recycling of body wastes causes the blood ratio of urea to creatinine (U/C) both expressed in mg/dl to decrease from 20 or more (sometimes ranging as high as 70 after eating a high protein diet) to 10 or less—something impossible for any other mammal that is not drinking fluid. A U/C ratio of 10 or less due to a significant decrease in urea and a significant increase in creatinine indicates that recycling of urea is in progress. The low U/C ratio found throughout denning sometimes occurs in wild bears in the fall just before denning. At this point, wild bears have stored enough fat for denning. They stop eating and drinking; complete waste recycling has begun before they enter the den.

The bear continues to degrade amino acids and form urea. In turn, the urea molecule is quickly degraded by transferring nitrogen from it to substances such as pyruvic acid or alpha-ketoglutaric acid to reform amino acids. This latter process is called transamination. The substances necessary for transamination (pyruvic acid and alphaketoglutaric acid) are generated from glycerol which has been released from fat. The newly formed amino acids are then reincorporated into protein.

The overall process of urea recycling consists of two processes: 1) formation of urea from amino acids, and 2) reformation of amino acids from urea which are then reincorporated into protein. Since (2) is faster than (1), there is net formation of new protein. Based on our knowledge, no other fasting animal can accomplish this feat.

Some amino acids formed in the bear are: alanine, serine, ornithine, arginine, glycine, leucine, threonine, phenylalanine, and tyrosine. These amino acids are found in such proteins as albumin and fibrinogen.

Humans can recycle only about 25% of the urea they form. The bear, on the other hand, recycles urea back into protein a little faster than it makes it. Thus, its blood urea concentration diminishes even though it does not drink water or urinate. The amino acids that serve as vehicles for urea recycling are ordinarily found in all mammals, but not in the concentrations shown by bears when fasting. Therefore, it is assumed that they may become vehicles to be used with the bear derived isolate when duplicating the bear's unique recycling.

During denning, the kidney of the bear continually forms urine. Upon reaching the urinary bladder, the urine (which contains BDI) is completely absorbed by the wall of the bladder. Thus, in a highly concentrated form, BDI moves across the bladder wall into blood, circulates, and stimulates all tissues of the bear. When compared to the blood of fasting humans, blood of the denning bear differs in concentrations of some amino acids, bear ketones are much lower, and there is a difference in some other essential substances. While concentrations of many of these substances decrease during human fasting; they do not decrease in the bear. Therefore, exact profiles of these known metabolites may have to be added to BDI in order to duplicate the bear's unique recycling in humans.

Recycling urea, the waste product of protein breakdown, back into protein leads to maintenance of lean body mass.

To prevent bone loss, bone remodeling occurs normally while in the supine state. In the human, a supine state inhibits normal bone remodeling and leads to severe loss of calcium and bone.

Bear derived isolate (BDI) which can be found in fasting and denning black bears which, in combination and with various carriers and various doses, based upon studies conducted with guinea pigs, bone cultures, and rats, will likely have beneficial results on humans in promoting bone growth in those persons having osteoporosis, in conserving nitrogen to a point where hemodialysis and kidney transplants need not be done in patients with chronic or end stage renal disease, in inhibiting protein breakdown in humans suffering burns and trauma, in permitting long-term flights into space by conserving bone integrity and preventing muscular atrophy, and in producing weight loss in obese subjects in the form of fat reduction while conserving lean body mass and promoting tranquility while in an alert state at normal body temperature.

A related aspect of the study of bear derived isolate (BDI) is directed to methods of the isolation and purification of the bear derived isolate, whether from a fasting bear or a denning bear, to a form where predictable results in the above phenomena are readily achieved alone or in combination with other known metabolic substances.

A better understanding of the field of invention, the invention itself, and the description of preferred embodiments will follow from an understanding of the definitions of various terms which are used, and which appear in the following “Glossary of Terms”.

GLOSSARY OF TERMS

Aliquot: A specified portion.

Alkaline Phosphatase Activity: Activity of this enzyme increases in bone as part of osteoblastic stimulation of bone growth.

Amino Acids: An amphoteric organic acid containing the amino group NH₂; any of the alpha-amino acids that are the main components of proteins.

Anorexia: Loss of appetite.

Aqueous Fraction: That portion containing water.

Bone Remodeling: A function of bone in which osteoblasts form bone and osteoclasts resorb bone. Positive bone remodeling occurs when the osteoblastic activity exceeds the osteoclastic activity; or when the osteoclastic activity is diminished; or where the osteoblastic activity is increased. In any of these events there is a positive addition to bone. Negative bone remodeling occurs when the osteoclastic activity outstrips the osteoblastic activity, or the osteoblastic activity is reduced from its normal balance with the osteoclastic activity; and any combination of the foregoing.

Bone Resorption: Occurs when bone is subjected to osteoclastic activity.

Countercurrent Chromatoratphy (CCC): A technique used to separate substances of different molecular characteristics by using solvents of aqueous and organic properties with centrifugation. Some substances are retained on the coil while others pass through.

Denning: To live or retire in a den.

Deproteination: Subject the sample to any of various procedures for removing all or part of the original protein in the sample.

Differentiation: To develop into specialized organs or cells.

Eluted: Drawn down, through or off (e.g. liquid through a filter).

Eluted Isocratically: Separate substances off of a column using one solvent system without changing concentration of that solvent system.

Fasting: A voluntary or involuntary state represented by states of non-ingesting, hypophagia, or anorexia. In the context of a fasting active summer bear, while food may be withheld, water is available on demand.

Fibroblast: A stellate or spindle-shaped cell with cytoplasmic processes present in connective tissue, capable of forming collagen fibers.

Gas Chromatography (GC): A method of chromatography in which the substance to be separated into its components is diffused along with a carrier gas through a liquid or solid adsorbent for differential adsorption.

High Performance Liquid Chromatography (HPLC): Method of partitioning chromatography that employs high pressures to propel the solvent through a thin column resulting in a high resolution of complex mixture.

Hyperphagia: Abnormally increased appetite for consumption of food.

Intraperitoneally: Inside the abdominal cavity.

Latin Square Design: An experimental design which gives statistical meaning to data when using small numbers of experimental units (e.g. numbers of animals, samples, etc.). The number of treatments tested is always equal to the number of experimental units being used and each experimental unit receives all treatments over time.

Lyophilization: The creation of a stable preparation of a biological substance or isolate (blood serum, plasma, etc.), by rapid freezing and dehydration of the frozen product under high vacuum.

Lyophilize: Freeze dry.

Mass Spectrometry (MS): A procedure used to determine the masses of atoms or molecules in which a beam of charged particles is passed through an electric field that separates particles of different masses.

Metabolites: Any of various inorganic or organic compounds produced by metabolic pathways in the body such as urea, creatinine, amino acids, hydroxy acids, fatty acids, glucose, ions, etc.

Monocyte: Cells with a single nucleus derived from marrow monoblasts. They have deeply indented and irregularly shaped nuclei and bundled and scattered single filaments in the cytoplasm. Marrow monocytes are responsible for forming osteoclasts.

Ninhydrin: Agent used to develop color on TLC plates.

Nuclear Magnetic Resonance (NMR): The absorption of electromagnetic radiation of a specific frequency by an atomic nucleus that is placed in a strong magnetic field, used especially in spectroscopic studies of molecular structure.

Osteoblast: A cell from which bone develops.

Osteoclast: A large multinuclear cell that resorbs bony tissue in the process of osteoclasis.

Osteoid: Relating to or resembling bone ossiform; newly formed organic bone matrix prior to calcification.

Osteoporosis: Demineralization of bone; decrease in bone mass or structure.

Ovariectomy: Surgical removal of the ovaries.

Pellet by Centrifugation: Spin sample to force protein and other residues to bottom of test tube.

Pharmaceutically Acceptable Carrier: Diluents and/or carriers, as described in Remington's Pharmaceutical Sciences, Arthur Osol, Ed., 16th Ed., 1980, Mack Publishing Company.

Peptides: Peptides are chains of amino acids. Two amino acid molecules are joined together by a linkage formed by removal of elements of water from one amino acid and the alpha amino group of another amino acid. This forms a dipeptide. Peptides of molecular weight up to 10,000 are known as polypeptides and above 10,000 as proteins. Peptides can be represented by various abbreviation symbols which are known to those skilled in the art. [Please see Lehninger Principles of Biochemistry by David L. Nelson and Michael M. Cox, 2005, published by W.H. Freeman and Company.]

Phosphomolybdic Acid Detection: Method used to develop color on TLC plates.

PWS: Prader-Willi-Syndrome—Prader-Willi syndrome occurs sporadically and is associated with fetal and infantile hypotonia, short stature; poor feeding in infancy but insatiable hunger later, leading to massive obesity; characteristic faces with almond-shaped eyes, small hands and feet after infancy, mental retardation and emotional instability in patients of either sex; delayed mearche in females; and micropenis and cryptorchism in males. Osteoporosis is common in these patients during the teenage years, and sex steroid replacement, when indicated, may help. Behavioral modification may improve the usual pattern of rampant weight gain. Patients have deletion or translocation of chromosome 15q11-13².

Renal Failure: Inability of kidney to function properly; one aspect is failure to excrete the amount of urea formed by the body daily. This leads to a gradual elevation of urea which may result in uremia, a toxic condition, that requires dialysis or kidney transplantation for treatment.

Resolution Factor (R_(f)): The distance that the midpoint of the compound travels on a given plate divided by the distance the solvent travels on the plate.

Resorb: To dissolve and assimilate.

Silica Gel/Column Chromatography: Sandlike material is placed in a long glass tube which is wet with solvents and is used to separate the materials by retaining some components on the silica while other components pass through depending on the solvents used.

Sham: A subject is subjected to surgical procedure without removal of organs (ovaries) in order to duplicate the physical and mental impact of the surgical procedure on test animals.

Silica Plate: Glass plate or microscope slide coated or painted with sand-like material. Used to separate and detect substances.

Stirring Rod: Metal or glass rod used to stir mixtures (e.g. spoon in coffee).

Supernatant: Liquid fraction of a liquid solid mixture where the solid has settled to the bottom of its container (e.g. in water and sand, water is the supernatant).

Therapeutically Effective Amount: An amount of a substance which attains a positive medical result.

Thin Layer Chromatography (TLC): Method used to separate chemical constituents which can then be identified by color or other properties upon development.

Transamination: A process involved in the metabolism of amino acids in which amino groups (—NH₂) are transferred from amino acids to certain keto acids yielding new keto and amino acids.

Triturate: Treat certain dry materials by dissolving part of them into solution leaving behind components that do not dissolve in said solution.

Ultrasonication: Using sound waves to remove particles from small places (e.g. used to clean jewelry).

SUMMARY OF THE INVENTION

The black bear alternates between annual states of hyperphagia and anorexia. In wild bears, hyperphagia begins in late summer. Becoming obese, many free-ranging bears have stored sufficient energy by early fall, stopping eating even when ample food was still available.

This behavior is seen in pregnant and solitary female bears as well as in male bears. Apparently, the bear is prepared for denning long before cold weather or considerable decreases in periods of sunlight occur. Coincident with this finding, the ratio of serum urea to serum creatinine is low, approximating values found early in denning. This low ratio reveals that the biochemistry for successful denning is operative.

The female bear is able to starve (in the sense of having no food or water intake) but carries out successful pregnancy and lactation. This situation combines three usually incompatible states: starvation, pregnancy and lactation.

Lasting 4 to 5 months, the state of anorexia achieved remains undisturbed. No odor is present and no urination or defecation has occurred.

Captive black bear studies have duplicated the anorexic phenomena. Bears remain healthy throughout the anorectic denning period from November to March despite not receiving food or water, as well as not urinating or defecating.

Prader-Willi Syndrome (PWS) is commonly designated as the H₃O syndrome: hypotonic musculature, hypomentia (mental deficiency), hypogonadism and obesity. Body composition studies support the concept of extreme obesity in people with PWS, with body fat content values as high as 53% of total body weight (Nelson et al., 1981).

In a study of patients with PWS, food intake was found to be over and above normal levels of intake with readings between 1000 and 2000 excess caloric intake. In fact, Bray et al. found that the average caloric intake of patients with PWS was 5167±503 calories a day.

A major contributor to the incidence of extreme obesity is the prevalence of early onset childhood hyperphagia in people with PWS, as reported in multiple publications (Grumback, M M and Styne, D M 1998; Bray et al., 1983; Nelson et al., 1981).

The primary basis for comparing the anorectic period experienced by bears with that of patients with PWS is that bears will suddenly stop eating while, once begun in the child with PWS, hyperphagia never stops.

The invention describes novel polypeptides which were isolated from black bear serum [Bear derived isolate]. These polypeptides are useful in appetite suppression. The basis of the invention lies in the identification and isolation of the specific polypeptides which are found in the BDI and are responsible for the anorectic state of black bears during the denning period. In the present invention human serum from subjects with Prader-Willi Syndrome (PWS) was compared with black bear BDI serum in order to identify and isolate the polypeptides responsible for the anorectic state of black bears but not present in the serum of the PWS subjects.

In a first aspect, the present invention features polypeptides useful in appetite suppression. These polypeptides are most preferably between about 12 and 13 amino acid residues in length and a mass (molecular weight as measured in Dalton) of about 1249.

In a second aspect, the present invention features a method of treating obesity by administering to obese subjects an effective amount of the polypeptides of the present invention.

In a third aspect, the present invention features pharmaceutical compositions comprising one or more of the polypeptides for appetite suppression. The preferred pharmaceutical compositions comprise an effective amount of one or more polypeptides of the present invention for appetite suppression. It is also envisioned that the preferred pharmaceutical compositions will also contain a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention features methods to isolate and to evaluate farther BDI which is present in the serum of the fasting or denning black bear.

Individuals with Prader-Willi syndrome suffer incessant appetite throughout their lives. In stark contrast, black bears annually cancel hyperphagia and quit eating beginning in November and lasting 100 days or more.

Although these contrasting states in bears have been known for years, it had been impossible to isolate the peptide or peptides responsible for appetite suppression. Comparing blood or serum of individual bears obtained under controlled conditions of hyperphagia and absent appetite has failed to isolate a unique peptide or peptides present during appetite suppression of bears and absent during hyperphagia.

Using cutting edge technology, which will be discussed later in this application, the inventors have now been able to isolate and identify the particular polypeptides responsible for appetite suspension in the bear.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1

Individual bear weights taken biweekly from October 2002 through October 2003.

FIG. 2

Weight percentages of bears taken biweekly for two years.

FIG. 3

Bear serum compared to pooled Prader-Willi serum.

DETAILED DESCRIPTION OF THE INVENTION

Methods used in this study relate to two newly discovered approaches. One is careful monitoring of annual weight and food intake changes in black bears as a measure for anorexia and hyperphagia. The other is use of cutting-edge technology which can be exemplified by Surface Enhanced Laser Desorption/Ionization (SELDI) and Matrix Assisted Laser Desorption/Ionization (MALDI). to identify and isolate specific peptides.

Animals

Thirteen captive adult American black bears (Ursus americanus) used in the study were maintained at the USDA approved Carle Foundation Bear Research Facility in Champaign County, Ill. All were unmated and physically isolated. The experimental protocol to obtain blood samples was approved by the Carle Foundation Hospital Institutional Animal Care and Use Committee. Bears were housed individually in covered indoor/outdoor enclosures and exposed to natural environmental conditions. Bears were maintained on generic dog chow or Sportsman's Choice®. Food was removed for the denning period from early November until mid-March each year, however water was still available ad libitum per USDA directive.

During spring, summer and fall active seasons, all bears were allowed food and water ad libitum. Not more than once a month, bears were anesthetized with Telazol®, 4-5 mg/kg body weight by intramuscular injection using either jab-pole or tranquilizing gun. Anesthesia was administered by the attending veterinarian from the University of Illinois at Urbana-Champaign Division of Animal Resources. Once anesthetized, bears were examined by a veterinarian and blood samples (3×60 cc) were drawn by femoral vein puncture. All bears were allowed to arouse from anesthesia and continue normal eating, drinking and/or denning patterns.

Bears were weighed without anesthesia using a specially designed and constructed mobile electronic weighing vehicle. This vehicle was rolled to the opening of the indoor cage, fixed to the cage so the bear could enter under its own will, be weighed and transferred back into its indoor enclosure. Weights were obtained biweekly, but, when necessary, taken weekly or daily (Goss et al, 2003).

Food intake was measured by weighing a specially constructed feeding container before attaching it to the cage and then weighing the sample container at a specific number of hours later.

Recruitment of Subjects with Prader-Willi Syndrome

Subjects were recruited through posters at national Prader-Willi Syndrome Association, 5700 Midnight Pass Road, Suite 6, Sarasota, Fla. 34242.—USA (PWSA-USA) meetings and an advertisement on the PWSA-USA website (www.pwsausa.org), as well as through word of mouth among participants and their friends and colleagues. The study protocol and recruitment procedures were approved at Institutional Review Boards at both the University of Illinois at Urbana-Champaign and Carle Foundation Hospital.

Subjects (or their legal representative, in the case of subjects who were minors) who signed an informed consent document were sent a kit containing all the materials needed to obtain a blood sample and send the sample back to the laboratory. Accompanying this kit was a questionnaire for the subject (or their legal representative) to fill out about early and current food behaviors.

Answers from these questionnaires were gathered and matched with the appropriate spectral data according to the subject's study number.

Analysis of Serum Samples

The SELDI-TOF ProteinChip® system (Ciphergen Biosystems, Fremont, Calif.) served as the means to initially separate, detect and measure the molecular mass of peptides directly from black bear serum and serum samples from persons with PWS. Serum peptides have an intimate association with food intake (Bray et al, 1983) and thus were chosen as a means for comparing bears and patients with PWS.

Crude serum samples were diluted with de-ionized water, then ultracentrifuged to deplete the samples of large proteins such as albumin and immunoglobulins.

The bear serum was processed on the Beckman Optima L, Rotor SW55Ti #1975 for 11½ hours at 55,000 RPM.

The Prader-Willi (human) serum was processed on the Optima, Rotor TLA 100.2 Tabletop, Beckman Ultracentrifuge for 178 minutes (3 hours) at 11,000 RPM.

The resulting supernatant was pretreated and dissolved in a matrix solution specific to the type of ProteinChip Array retentate chromatographies used (weak cation exchange on WCX2, strong anion exchange or SAX2, or hydrophobic/reversed phase or H4), as outlined in the ProteinChip User's Guide. Once the sample is bound to one of the 8 spots available on each ProteinChip, an Energy Absorbing Molecule (EAM) solution is applied, which forms a crude crystal when dry. Each chip with sample was placed into the ProteinChip reader, which aims a laser at each ProteinChip spot. The energy from the laser, combined with a voltage differential, allows the sample to become gaseous and “fly”. The time of flight is recorded and, from this measurement, the ProteinChip analysis software can calculate the molecular mass. The collection of molecular masses provided a spectrum of proteins and peptides that appeared in each sample.

The resulting spectra were analyzed for peaks of interest. Peaks that only appeared in spectra from bear serum collected in November and not in PWS spectra were preliminarily identified by their molecular weights from the ExPASY Tag IDENT database and compared to human substances in the Scan Swiss-Prot database.

These unique peptides were compared with entries in the Scan Swiss-Prot database to identify specific peptides to submit for sequencing. They were also compared to a list of established compounds (Table A) proven to suppress feeding.

TABLE A Peptide Mass (Da) Reference Cyclo-His-Pro 234.3 Sigma Catalog Thyrotropin Releasing Hormone 354.4 Sigma Catalog Enterostatin 496.6 Obesity Res.; 5,360 (1997) Cholecystokinin-4 (CCK-4) 596.7 Sigma Catalog Oxytocin 1007.2 Sigma Catalog Vasopressin 1084.2 Sigma Catalog Cholecystokinin-8 CCK-8 1143.3 Sigma Catalog Cholecystokinin-12 CCK-12 1535.7 SwissProt Database Bombesin 1619.87 Merck Index Neurotensin 1672.9 Sigma Catalog Cholecystokinin-20 (CCK-20) 1937.4 SwissProt Database Urocortin UCN3 2304.97 SwissProt Database Glucagon 3358.7 Sigma Catalog Calcitonin 3417.9 Sigma Catalog Cholecystokinin-33 (CCK-33) 3945.5 SwissProt Database Urocortin III (UCN3) - human 4138.9 SwissProt Database PYY (human) 4310.8 SwissProt Database Cholecystokinin-39 CCK-39 4626.27 SwissProt Database Corticoptropin Releasing 4757.5 Sigma Catalog Hormone (CRH) Insulin (human) 5807 Sigma Catalog

Compounds proven to suppress appetite in humans. Matrix Assisted Laser Desorption/Ionization Mass Spectrometry—Time of Flight (MALDI-TOF; Ciphergen Biosystems, Fremont, Calif.), a technology similar to SELDI-TOF, was used for sequencing peptides identified by the SELDI-TOF analysis.

Results and Discussion

Anorexic Bears

Food was withheld beginning on Oct. 31, 2002, after careful observation of the captive bears revealed no obvious signs of hunger such as ransacking behavior, rummaging efforts, searching behavior or indications of nervousness related to the absence of food. Bears were left alone in the tranquil nature for the next 4.5 months, except when disturbed to obtain blood samples and biweekly body weights.

Body weight changes are depicted in FIG. 1. The anorectic period lasted from Oct. 31, 2002 through Mar. 21, 2003. Individual responses of each of the 12 bears were remarkably similar in pattern. When data were expressed as percent changes in body weight over a 2-year period, a striking reproducibility of body weight changes is highlighted (FIG. 2).

During the anorectic period, which usually occurs between November and early March, the body temperature of the bears demonstrated a significant, but extremely small decrease from a normal value of 36.77° C. to 36.14° C. (98.18° F. to 97.05° F., p<0.001) (Nelson, 2001). The ratio of serum urea to creatinine also remained low during this period, having values well below those of non-fasting months (FIG. 2).

Bears became active in early March and were allowed food ad libitum. Entering a period of hyperphagia, male bears ate an average of 9000-10,000 kcal/day. Body weight steadily increased until early May.

Food intake then decreased to summertime low levels in May and June, but a stable state of body weight gain was finally acquired by mid July to early August.

In mid August, a second state of hyperphagia began. By September, food intake reached a maximum with male bears eating an average of 19,381 kcal/day. This trend lasted into October where body fat storage finally became sufficient to support over-winter denning and an extended period of anorexia.

Feeding History of Persons with Prader-Willi Syndrome

Questionnaires were returned for 20 of the 22 subjects who submitted a serum sample. In the questionnaire, subjects (or their parent, guardian or legal representative) reported feeding history from the first few months of life as well as current eating behaviors.

Early Feeding History

Of 19 total respondents to the questions regarding early feeding history, 13 reported that the person with PWS had to be awakened to be fed; three for 6 weeks or less, six for 6 weeks to 6 months, and four participants had to be awakened for over 6 months.

Eight respondents reported that the subject demonstrated “appropriate” feeding schedules at less than 1 year of age. Another eight respondents reported that the subject was over the age of 1 year when their feeding schedule became “appropriate”.

Thirteen respondents indicated that the subject had to be gavage fed; 10 for under 6 months and 3 for over 6 months.

Current Feeding History

Of seven minor subjects (ages 3-35 months), five reported eating behaviors to be appropriate for their age group while two subjects reported above average (eager or hyperphagic) eating behavior that began at ages 3 and 27 months.

Of four slightly older minor subjects (ages 4-7 years), all reported hyperphagic behavior beginning around age 2 (range 18 months-3 years). Three out of the four subjects also reported gorging, stealing and hoarding behaviors while it was indicated that the remaining subject, while “always hungry”, was on a strict, parentally controlled diet.

The remaining nine subjects (adults, aged 14-33 years) also reported current hyperphagic behavior. Five of the nine subjects reported gorging behavior and six reported stealing food.

Several subjects from each age group reported that food in refrigerators and cabinets was locked up at all times.

Peptide Profiling—Anorexic Bears vs. PWS Subjects

A summary of SELDI-TOF spectral data revealed 359 peaks isolated in PWS subjects younger than 36 months and 376 peaks in subjects between the ages of 36 months and older, for a total of 735 peaks; 291 of these 735 peaks were shared between both age groups.

The objective of the research was to determine if unique peptides are present in the denning bear that are responsible for appetite suppression. By virtue of the ability of SELDI-TOF determinations to isolate denning bear (November) serum findings from findings from serum of hyperphagic PWS subjects, called the “subtraction technique”, profiling of peptides was taken to a second level.

The subtraction technique—allows the inventors to isolate 75 peptide substances in the November serum not represented among the spectral peaks of substances found for the PWS serum. The mean ‘molecular weight’ of the measured distribution of these 75 substances was 2225.1538±506 Da (where 1 Da, or Dalton, can be considered equal to 1/12th of the weight of an atom of Carbon-12). This mean weight can be found by determining each of the ‘relative amounts’ and the individual ‘dalton weights’ of each of the 75 distinct substances, then summing the products of each of these pairs of relative amounts and weights for each of these 75 peptides. This sum is the mean molecular weight.

A standard error (SE) for the distribution (the individual fractions) of these distinct peptides can be found by squaring each of the numbers (residuals) that result after subtracting the mean molecular weight from each of the 75 individual Dalton weights, multiplying these 75 squared residuals by their respective fractions in the spectrum, and forming the sum of these 75 new values. The SE is the square root of this final sum. (The applicants have obtained the value SE=506.)

The applicants then decide that if they double this SE value and add and subtract it from the mean molecular weight value, they will get a 2 SE range or interval of ‘likely’ Dalton weights within which the unique peptide's weight should reside. Thus, the applicants determine this range is 1213 to 3237 Da.

The applicants then identify a ‘unique’ peptide in the observed spectra that has:

-   -   a weight in the above range; and also,     -   is near the weight of a peptide among a set of peptides known or         reputed to exhibit ‘appetite suppression protein’         characteristics (see Table A).

When the results from the subtraction technique were compared, 75 peptides from November serum were culled out, representing 75 substances not found in the 735 peaks from PWS serum. The mean of the mass spectroscopic distribution of these 75 substances (or unique peptides) was 2225.1538±506 Da. The range of distribution was taken at ±2 SE or between 1213-3237 Da. Thus, the candidates for unique peptides was reduced to 16 possibilities.

The next issue became how many of the 16 remaining peptide candidates would be suitable for sequencing. Only one peptide, at a mass of 1249.69 Da, was identified as specific only to November bear serum (FIG. 3). When compared with established peptides associated with appetite suppression (such as those listed in Table A), this unique peptide was closest to Cholecystokinin-8 (CCK-8). The effects of CCK-8 have been found to be primarily related to the suppression of food intake in humans.

This peptide was also found to be unique by a search of the Scan Swiss-Prot database. The closest candidate found in the search of over 300,000 entries was a protein weighing 1251 Da and not related to food suppression.

A MALDI-TOF analysis was performed on the unique peptide and, surprisingly, two amino acid sequences were found for the same mass of 1249.69. These amino acids are represented by the following formula:

RTGEGFLVTGGGV (SEQ ID NO: 1) VSKEGFLVTGGR. (SEQ ID NO: 2)

When compared with the 3 published amino acid sequences (Sigma Chemical Catalog) for CCK-8, little repetition was found.

The singular finding of two distinctive peptide sequences for the same mass as the prime indication of a peptide involved in feeding is both reproducible and unique. The fact that it is limited to the November anorexic bear and not found in the hyperphagic PWS subject, and that it is similar in the range of mass to the CCK-8 peptide, warranted it suitable to be considered an “appetite suppression protein” and tentatively named “1249 peptide”.

The present applicants have discovered methods for isolating and evaluating the various components or compounds found in the serum of the fasting and denning black bear. The results of those discoveries were present in applicants' previous applications which were detailed earlier in the specification and are now represented.

Applicants invention results from isolation of a material in bears, particularly black bears, called Bear Derived Isolate or BDI, that enables denning so that BDI can be used alone or identified with one substance or combination of substances either novel and unique or previously identified to help human beings and other mammals. All predictable results are based upon in vivo studies with guinea pigs, in vivo studies with rats, in vitro organ studies of calvarial mouse bone, and in vitro studies of prevention of proliferation of cells that resorb bone and stimulation of proliferation of cells that form bone using cell cultures of monocytes, osteoclasts, osteoblasts and fibroblasts. BDI is present in the serum (blood) of denning bears. BDI is also present in urine of denning bears. However, because the bear is an omnivore, fasting in summer is extremely rare. What has been discovered however, is that when the normally active black bear is fasted in the summer time, but water not withheld, over a period of two to three weeks it will develop in the urine the same BDI referred to with regard to denning black bears. Post-fast data showed that urea recycling was induced. This was evidenced by a low serum urea/creatinine ratio, a slight increase in total proteins, and a marked increase in beta-hydroxybutyric acid. Accordingly where the term BDI is used, it includes fasting bears from which food has been withheld but which are not in the traditional denning season. The same can be extrapolated for active polar bears. Because the U/C ratio of polar bears is near 10 or less when fasting, urea recycling is indicated.

In order to obtain the research material (BDI) blood (serum) and urine are collected from black bears during their denning period. Quantities of 100 ml may be drawn monthly from each bear or on a more frequent schedule as required. The urine and/or serum is then subjected to the isolation method as described herein.

As illustrated in Table 1, isolation of BDI requires precipitation of protein from winter urine or serum using methanol, centrifuging the sample and removing precipitated protein as pellets, and drying the BDI into a visible extract. Further, by the use of thin layer chromatography (TLC), countercurrent chromatography (CCC), preparative thin layer chromatography, or column chromatography, at least two compounds, both in urine and blood, can be isolated in BDI.

Thus, the method of isolating these compounds permits predictable separation of BDI into Fractions. These Fractions are suitable for biologic testing. One component is an as-yet-unidentified compound. It is called the Miers-Nelson Component (MNC) after the researchers. The other component is beta-hydroxybutyrate (BHB).

BDI can be divided into three Fractions which are sufficiently purified to test for their biological activity in guinea pigs, rats, and bone culture assays. These Fractions are:

-   -   Fraction I=BDI-[BHB+MNC] (Early fractions),     -   Fraction II=BHB (Middle fractions), and     -   Fraction III=MNC (Late fractions).

Following is a Table illustrating the process for the isolation of BDI and two compounds found in it.

TABLE 1 Chemical Process for Isolation of BDI And Two Compounds Found In It Research Procedure for Isolating BDI and Its fractions STEP SAMPLE PROCESS YIELDS One Urine (50 ml) 1. MeOH Deproteinization Dry Sample (BDI) 2. n-BuOH Trituration Two Dry BDI (3.5 g) CCC (n-BuOH:AcOH:H₂O) Dry Sample 2-:1:20 Three Dry sample (2 mg) CCC (n-BuOH:AcOH:H₂O) Fractions: 2-:1:20 A. Fraction I BDI − [BHB + MNC] Early CCC Fractions B. Fraction II BHB Middle CCC Fractions C. Fraction III MNC Late CCC Fractions THE DENNING PROCESS OF BEARS

The denning process of bears has been defined in the statement of Background of the Invention above. In order to obtain the bear derived isolate successfully, denning bears must be available quickly and throughout the denning period as is the case at The Carle Foundation Bear Research Station, Champaign County, Ill. At this facility, after food intake decreases in October or November, food is removed, inducing the bear to enter the denning state. At all times where reference is made to the bears which were used to produce BDI, such bears were the well known North American Black Bears (Ursus americanus).

Thereafter, blood and urine samples are taken from the bears. This continues until March when the bear leaves its den and has access to food and water. At first (for approximately two to three weeks), the bears slowly begin to eat after they emerge from their dens in the spring. Food intake reaches normal levels, and weight gain continues until early June in preparation for mating. By mid June the bears have normalized their body stores of fat that were diminished during denning and will continue to eat throughout the summer to maintain body weight. Slight increases in body weight throughout the summer can be attributed to continued growth. In late August, in preparation for the subsequent denning season, the bear increases its food intake from 5,000 to 8,000 Calories/day to 20,000 Calories/day. The bear eats almost to a calorie the quantity of food required to store enough fat to support energy requirements of denning, fetal support, and lactation. For a 400 pound bear, energy expenditure during denning is about 4,000 Calories/day.

Bears that have been fasted for a period of not less than 21 days during the summer or non-denning period, whose urine, when subjected to isolation methods, yielded a material (BDI) which produced bone remodeling effects and urea creatinine ratios comparable to that of the material (BDI) taken from a denning bear. The experiment related to 14 bears which were given free access to drinking water, but food was withheld for 21 days. The group was fasted during the month of July, a recognized non-denning period for bears. This was in an attempt to determine whether fasting is the controlling factor in the production of BDI.

Defecation stopped after approximately 2-3 days in the fasting bears, but occasionally bile stain material passed per rectum in some of the bears. With free access to water, the bears drank enough to stimulate urination. (Excess water was required because the only mechanism bears have to regulate body temperature is through evaporation via the respiratory tract. In summer, ambient temperature is much higher than experienced by denning bears, thus there is a need for increased evaporative water loss. This, in turn, stimulated drinking, which exceeded the bears' requirements for body temperature control and thus stimulated urination.) Even though the fasted bears drank water, thirteen of fourteen bears showed an increase in serum creatinine. Eleven of fourteen bears showed a reduction in serum urea, which resulted in a significant reduction in the U/C ratio. Five animals demonstrated values previously known to be associated only with denning bears (Table 2).

TABLE 2 SUMMER BEAR FASTING EXPERIMENT: Jul. 13, 1994 to Aug. 2, 1994 DATE Jul. 13, 1994 to Jul. 13, 1994 Aug. 2, 1994 Aug. 2, 1994 Jul. 13, 1994 Aug. 2, 1994 Jul. 13, 1994 Aug. 2, 1994 Jul. 13, 1994 Aug. 2, 1994 PRE-FAST POST-FAST WEIGHT PRE-FAST POST-FAST PRE-FAST POST-FAST PRE-FAST POST-FAST WEIGHT WEIGHT LOSS UREA UREA CREATININE CREATININE U/C U/C BEAR (lbs.) (lbs.) (lbs.) (mg/dl) (mg/dl) (mg/dl) (mg/dl) RATIO RATIO 1-524 256 214 −42 22.39 21.89 1.4 2.1 15.99 10.42 2-523 186 150 −36 29.61 36.70 1.4 2.2 21.15 16.68 3-519 358 298 −60 31.70 27.47 1.7 2.6 18.65 10.56 4-521 226 186 −40 32.60 41.85 1.7 2.1 19.18 19.93 5-522 350 302 −48 30.90 18.24 1.8 2.1 17.17 8.69 6-520 298 248 −50 32.20 30.90 2.1 2.4 15.33 12.88 9 7-513   210 178 −32 30.70 26.61 1.5 2.1 20.47 12.67 9 8-514   216 190 −26 45.50 27.47 1.7 2.6 26.76 10.56 9-515 306 260 −46 37.98 30.26 2.2 2.3 17.26 13.16 9 10-516   162 140 −22 33.00 31.55 1.6 2.2 20.63 14.34 11-518  304 262 −42 19.74 36.48 1.6 2.6 12.34 14.30 12-517  306 260 −46 44.40 24.46 2.3 2.0 19.30 12.23 U.P. 412 356 −56 49.35 24.46 2.4 2.7 20.56 9.06 Caruso 388 328 −60 42.30 31.76 1.9 2.4 22.26 13.23 MEANS 284 ± 77 241 ± 67* −35 ± 15 34.46 ± 8.5 29.29 ± 6.3 1.8 ± 0.3 2.3 ± 0.2 19.08 ± 3.47 12.75 ± 3.0* SUMMARY Active bears eating normally with fasted 21 days. After fasting: 1 11 out of 14 bears showed a decrease in the concentration of serum urea. 2. 13 out of 14 bears showed an increase in serum creatinine. 3. 12 out of 14 bears showed a decrease in the U/C ratio with 5 bears showing values ≦10. *Indicates a significant difference between the Pre-fasting and Post-fasting values using a paired t test, p < 0.01.

Data collected from fasted summer bears (after they had been eating normally during the non-denning period) were compared with data collected from fasted winter bears. Although bears usually den (and don't eat) during the winter, these bears had been eating prior to entering the Carle Bear Research Facility. The data collected from fasted summer bears were similar to data collected from the same bears after a three week winter fast (Table 3).

TABLE 3 WINTER BEAR FASTING EXPERIMENT: Feb. 14, 1994 to Mar. 7, 1994 DATE Feb. 14, Feb. 14, 1994 to Feb. 14, Feb. 14, 1994 Mar. 7, 1994 Mar. 7, 1994 1994 Mar. 7, 1994 1994 Mar. 7, 1994 Feb. 1, 1994 Mar. 7, 1994 PRE-FAST POST-FAST WEIGHT PRE-FAST POST-FAST PRE-FAST POST-FAST PRE-FAST POST-FAST WEIGHT WEIGHT LOSS UREA UREA CREATININE CREATININE U/C U/C BEAR (lbs.) (lbs.) (lbs.) (mg/dl) (mg/dl) (mg/dl) (mg/dl) RATIO RATIO 1-524 280 230 −50 15.02 10.73 1.5 2.0 10.01 5.37 2-523 192 156 −36 17.17 19.31 1.6 2.2 10.73 8.78 3-519 384 332 −52 30.04 15.02 2.1 2.7 14.31 5.56 4-521 288 238 −50 32.18 12.88 1.7 2.1 18.90 6.13 5-522 380 324 −56 19.31 15.02 1.7 2.3 11.36 6.53 6-520 282 244 −38 23.61 10.73 2.2 2.5 10.73 4.30 9 7-513   228 206 −22 27.90 10.73 1.8 2.1 15.50 5.11 9 8-514   222 198 −24 36.48 21.46 2.2 2.4 16.58 8.94 9-515 328 282 −46 32.19 32.19 2.2 2.3 14.63 14.0 9 10-516   184 152 −32 27.90 27.90 1.6 1.8 17.44 15.50 11-518  318 286 −32 32.19 21.46 2.4 2.9 13.41 7.40 12-517  354 316 −38 17.17 10.73 1.5 2.0 11.44 5.36 *U.P. 380 374 −06 10.73 10.73 3.3 3.4 3.25 3.16 *Caruso 436 426 −10 6.40 6.44 3.2 3.2 2.01 2.01 MEANS 286 69 247 ± 62 −43 ± 15 25.88 ± 7.19 17.30 ± 7.21** 1.9 ± 0.3 2.3 ± 0.3** 13.72, 2.92 7.73, 3.57** SUMMARY Of the bears who were not previously denning (i.e., had access to food during the winter), after fasting: 1 9 out of 12 bears showed a decrease in the concentration of serum urea. 2. 12 out of 12 bears showed an increase in serum creatinine. 3. 12 out of 12 bears showed a decrease in the U/C ratio with 10 bears showing values ≦10. *Bear was already denning. **Indicates a significant difference between the Pre-fasting and Post-fasting values using a paired t test, p < 0.01,

It was concluded that after both the summer fast and the winter fast, the bears were in the urea recycling mode previously only characterized during denning.

The prefasted BDI from summer urine tested in bone cultures was from catheterized specimens while the post BDI from urine was collected without anesthesia from the specially adapted metabolic cages. As described later, BDI from the latter sample significantly increased osteoblast activity.

CHEMISTRY OF THE INVENTION

Introduction

The presentation to follow is divided into two parts. The first deals with the chemical process of isolation and characterization of BDI and two compounds characteristic of the winter denning bears (BHB and MNC) found in BDI. The second part describes the biologic activity of BDI and three of its component Fractions. The chemical isolation of BDI using chromatography makes it possible to divide purified BDI. Countercurrent chromatography yields 50 fractions in successive order: 1-50. The first group of CCC fractions (1-17) does not contain either BHB or MNC. The second group of CCC fractions (18-22) contains BHB. The third group of CCC fractions (23-50) contains MNC, found mainly in fractions 25-29. The CCC machine is then washed out to collect anything left in it. The third division also includes the wash; nothing is discarded. CCC fractions are grouped for further studies and labeled Fraction I, Fraction II, and Fraction III.

The specific fractions related to CCC samples may vary slightly. For instance, BHB may elute in fractions 19-23, and MNC in fractions 24-29. However, all CCC samples at division points are tested by thin layer chromatography so that no BHB appears in either Fraction I or Fraction II and so that no MNC appears in Fraction II.

Therefore, through the use of CCC, two characteristic components can be isolated. They also serve as logical points for division of BDI into three Fractions in order to test biologic activity: Fraction I (BDI-[BHB+MNC]), Fraction II (contains BHB), and Fraction III (contains MNC). When separated by CCC, these Fractions are known to contain amino acids, ammonia, urea, creatinine, creatine, and other animal products.

Identification of Bear Derived Isolate (BDI) Derived from Urine

A 50 ml aliquot of bear urine is deproteinated by diluting with methanol (1:1 v/v) and allowing proteins to precipitate out overnight at −20° C. The proteins are then pelleted by centrifugation (20 minutes @ 2500 r.p.m., 10° C.) and the supernatant is extracted. To completely dry the supernatant extract, nitrogen gas is used to remove methanol. Samples are then frozen (−80° C.) and lyophilized. Once dry, samples are weighed using Mettler Analytical Balance AE163. Fifty milliliters of winter bear urine yields approximately 3.5 g of dry residue known as BDI. For observation of the effects of BDI, the dry deproteinated sample (BDI) is reconstituted with 2 or more mL of saline. This solution can then be used for guinea pig and bone culture studies.

Isolation and Characterization of the Miers-Nelson Component (MNC)

Step I: Verification of MNC Presence in BDI

BDI containing MNC is prepared as before and dried to a residue using nitrogen gas or lyophilization. The BDI is then:

-   -   Dissolved in 100-500 μl of methanol depending on sample weight.

To test for presence of MNC in number (1) above, approximately 4-6 μl is applied to normal phase TLC plates (EM Science, P.O. Box 70,480 Democrat Road, Gibbstown, N.J. 08027-1296 Silica Gel 60 F₂₅₄₉ 0.25 mm) in successive, μl applications.

The silica plate is then developed in a 4:1:1 1-butanol:acetic acid:water solvent system contained in a TLC chamber. Once developed, the plate is removed, dried by heat gun, and finally detected by ninhydrin spray (0.3% w/v in 1-butanol).

Location of MNC is detected with vigorous heating by heat gun and/or hot plate until edges of the TLC plate are charred.

At this point in isolation, MNC is visualized as a pink spot at R_(f)=0.74-0.80.

Step II: Purification of MNC

Approximately 1.75 g of BDI containing MNC is then prepared for the next purification step involving countercurrent chromatography. This procedure utilizes a bi-phasic solvent system of 1-butanol:acetic acid:water (20:1:20) and a Countercurrent Chromatography System with #10 semi-preparative coil (P.C. Inc.).

Two liters of the bi-phasic solvent described above is prepared at least one day prior to using CCC.

This butanol-acetic acid-water solvent system is mixed by shaking and allowed to settle 2 to 4 hours before separation of the organic and aqueous bilayers.

Two liters of solvent yields approximately 1200 ml of the organic stationary phase (primarily composed of butanol) and approximately 800 ml of the aqueous mobile phase (primarily composed of water).

The dried sample of BDI that has been prepared prior to the aqueous/organic solvent system still contains MNC. This sample is reconstituted in 5 ml of the solvent system (2 ml stationary phase:3 ml mobile phase) and loaded on to a 10 ml injection loop interfaced to the CCC.

The CCC coil is first loaded with 385 ml of stationary (organic) phase.

Using the mobile (aqueous) phase, the triturate is injected onto the coil for separation.

The coil is rotated at approximately 80° r.p.m., flow rate=4 ml/min (LDC Analytical Mini Pump). Five minute samples are collected (Gilson Microfraction Collector #203).

Fifty (20 ml) samples are collected and the coil is washed with methanol:water (1:1 by volume).

All samples are then frozen (−80 C) and lyophilized (freeze dried).

Once dry, the 50 samples are analyzed by TLC/ninhydrin to determine which samples contain MNC.

MNC elutes in samples 25-29 (approximately 520-580 ml post coil).

Next, those usable, isolated MNC samples are combined with each other for further purification. Sample weight at this stage of purification has been reduced from 1.75 g to 1-2 mg. At this point, samples containing concentrated MNC also contain biological salts and significantly reduced concentrations of other impurities as detected by TLC/ninhydrin, UV, iodine vapor, and phosphomolybdic acid.

Then, samples containing MNC, the remainder of the CCC samples, and the wash of the CCC (fractions 22 through 50 plus wash) are recombined and passed through CCC a second time under the exact conditions described above.

Step III: Harvesting MNC: Preparative Thin Layer Chromatography

Final purification of Fraction III (MNC) entails the use of preparative thin layer chromatography.

The dried combined samples of MNC from the second countercurrent chromatography run are the sources of samples to be applied across an 8×12 cm silica thin layer plate. MNC is first reconstituted in 100 μl of methanol and then applied in ten 1 microliter (μl) spots across the plate.

Application of MNC in solution (to the TLC plate) is then repeated 10 times. In order to achieve the best resolution, between each application the μl spots are allowed to air dry. When finished, each spot on the plate will contain 10 microliters (μl) of MNC in solution forming a band across the TLC plate.

The plate is then resolved in 4:1:1 BuOH:AcOH:H₂0. Once the solvent rises to 80%-90% of the TLC plate, the plate is removed from the solvent and dried by heat gun.

Without developing the plate, the MNC band is removed by scraping the silica from the plate at the R_(f) region of 0.74-0.80.

The silica is then wetted in approximately 1-2 ml of 1-butanol with vigorous vortex mixing.

The 1-butanol and silica mixture is then centrifuged for 20 minutes at 2500 r.p.m. This allows the silica to pellet to the bottom of the tube.

The MNC containing butanol supernatant is then removed and dried down under nitrogen gas.

At this step in purification, the 1-2 mg sample has been reduced to 100-200 μg of MNC and is separated from salts and other impurities as detected by TLC/UV, ninhydrin, and iodine vapor. A lipid contaminant is apparent under phosphomolybdic acid development at the solvent front of normal phase TLC plates at this point. However, MNC remains the only significantly concentrated material present as detected by TLC/ninhydrin, UV, iodine vapor, and phosphomolybdic acid detection.

Properties of MNC

The harvested MNC has the following properties:

-   -   1. It is soluble in water, methanol, and 1-butanol.     -   2. It is insoluble in less polar organic solvents such as         chloroform, toluene, and hexane.     -   3. It is stable when stored frozen at −20° C. to −85° C. for at         least eight years.     -   4. It is stable at room temperature (20° C.-22° C.) for at least         four days.     -   5. It is heat resistant to 65° C.     -   6. It is slightly UV active by detection of TLC and UV         spectroscopy at 280 and 320 nm wavelengths.     -   7. It is ninhydrin positive only with extended heating as         previously described.     -   8. It can be identified as pink in color at R_(f) 0.77-0.80 when         purified on normal phase silica TLC plates, sprayed with         ninhydrin and heated.     -   9. It can be detected using iodine vapor development of normal         phase silica TLC plates.     -   10. To date, no tested substances in blood and urine of mammals         show characteristics similar to the ninhydrin reaction at R_(f)         range of 0.77-0.80 on the thin layer chromatography used in         isolation.     -   11. Recommended storage of the harvested MNC is to freeze it in         a light resistant container under nitrogen gas.         Isolation and Characterization of Beta-hydroxybutyric Acid (BHB)         Preparative Thin Layer Chromatography

The verification, purification, and harvesting of BHB is similar to MNC, except that CCC samples 18-22 are used to elute BHB. Further, BHB is extracted using the same method of preparative thin layer chromatography except that the silica is scraped from the plate at the R_(f) region of 0.82 to 0.92.

Flash Column Chromatography

An alternative method of harvesting BHB called Flash Column Chromatography can be used. When this method is used, BHB samples obtained from CCC purification are combined and dried.

The combined samples are reconstituted in 250 μl of 1-butanol. Mixing and ultrasonication are used to induce the sample into a homogeneous solution.

Once the samples are completely solubilized in the 250 μl of butanol, 250 μl of acetone is added to the solution. The resultant 500 μl sample is ready for subsequent purification by silica gel flash column chromatography.

A 15×230 mm silica gel (0.040-0.063 mm particle, 230-400 mesh) column is packed and wetted with five column volumes of acetone: 1-butanol (99:1). This ratio significantly contributes to purity and yield.

The 500 μl samples, in 1-butanol:acetone (1:1), are applied to the column and are desirably eluted isocratically with acetone: 1-butanol (99:1) under nitrogen gas pressure (5 psi) at a rate of approximately 2 in/mm. Fifty (1 ml) samples are collected in approximately 20-30 minutes.

Since acetone is the primary solvent, all collected samples are dried by nitrogen gas or allowed to air dry, and then visualized by TLC/ninhydrin. BHB elutes off the column in samples 19-21 with good reproductibility and resolution given the method employed.

SUMMARY OF PREPARATION OF PRE-FASTED AND FASTED URINE

The bears were fasted overnight before the day of the experiment. They were allowed unlimited access to water. On the day of the experiment bears were anesthetized with Telazol, i.m. 4-5 mg/kg body weight. Baseline blood and urine (catheterized) were taken as pre-fast controls. Catheterized urine was only collected from three of the bears, numbers 4/521; 9/515; and 12/517. The urine was pooled and treated with an equal amount of methanol (165 ml). After sitting overnight at 0° C., the urine was centrifuged at 1650 gravity×15 minutes. The supernatant was removed and the precipitate discarded.

Next, the supernatant was placed under a nitrogen stream until most of the methanol had been removed. The sample was then frozen at −80° C. After freezing, the sample was placed on the lyophilizer. YH 11-9-1 (BDI-U) was then used either for use in the bone culture or further purification by countercurrent chromatography (CCC).

Twenty-one days later, the bears were again anesthetized to collect serum and urine in the same fashion as the pre-fasted controls. Prior to this, beginning Jul. 28, 1994 until Aug. 2, 1994, urine was also collected from beneath the cages. All male urine was pooled and female urine was pooled. Catheterized urine was collected from bears and kept separately and treated with an equal volume of methanol after aliquots were removed for urea and creatinine analysis: 6/520 (4 ml, YH 11-13-2), 9/515 (119 ml, YH 11-13-3), and 11/518 (17 ml, YH 11-13-4). Also collected from two of the older bears was 125 ml from Caruso (YH 11-13-5), and 6.5 ml from UP (YH 11-13-6).

The samples were purified by countercurrent chromatography in the following manner. The dried, deproteinated serum (BDI, 0.5 to 1.0 g), was reconstituted in three to four ml of a lower phase 1-butanol:acetic acid:water (20:1:20) mixture. Ten fractions were collected in one run according to the standardized protocol (as attached). The samples were then lyophilized, reconstituted in methanol for transfer to pre-weighed vials, and then dried down under nitrogen for weight determination. At this point, samples were then evaluated for further bone cultures, lc/ms or further purification by HPLC. The cultures which were run with urine produced enhanced bone remodeling both of the osteoblastic enhancement and the osteoclastic diminution.

Formation of the Organic Bone Matrix-Osteoid

Both osteoblasts and fibroblasts are involved with formation of osteoid, the matrix of bone. BDI directly stimulates proliferation of osteoblasts, increasing their numbers by 129%. In a similar fashion, BDI directly stimulates proliferation of fibroblasts by 205%. BDI was tested in fibroblast cultures of NIH-3T3 cells. The concentration of BDI that achieved maximum results was 10 mg/ml, the same concentration that achieved maximum results in the osteoblast cultures of MC-3T3 cells. Thus, BDI coordinates the final stage of bone remodeling by furnishing a place to put new bone. BDI induces a similar significant proliferation of fibroblasts (the cells that form matrix or osteoid), the supporting structure of bone, as BDI induced in osteoblasts. Furthermore, the proliferation response of fibroblasts to BDI is similar to proliferation and the bone production response of osteoblasts to BDI.

Thus, BDI orchestrates bone remodeling in a remarkable fashion. In order to form bone while under the combined stresses of not eating or drinking, remaining non-weight bearing, and in the absence of sex steroid production, the bear makes enough bone to avoid osteoporosis. To do this, the bear must shut down bone resorption, stimulate bone formation, and prepare a place to put the newly formed bone. The bear accomplishes this by inhibiting bone resorption while simultaneously stimulating bone formation.

Vitamin D and Bone Integrity in the Denning Bear

During denning, unopposed action by the active form of vitamin D, 1,25-dihydroxyvitamin D₃ would produce bone loss, high blood calcium, and death. Ordinarily, 1,25-dihydroxyvitamin D₃ stimulates the gut to absorb calcium to replace calcium lost in urine. If insufficient calcium is in food, 1,25-dihydroxyvitamin D₃ stimulates bone to release calcium (bone resorption) to keep blood levels of calcium constant.

Since the denning bear is fasting and not urinating, unopposed action of 1,25-dihydroxyvitamin D₃ on bone would constantly stimulate bone to release calcium, causing blood calcium to rise to high enough levels to cause cardiac standstill and death. To prevent this occurrence, the bear reduces production of 1,25-dihydroxyvitamin D₃ while increasing production of another form of vitamin D—24,25-dihydroxyvitamin D₃. Considered by most a metabolite of vitamin D that has no metabolic action and normally excreted from the body, the 24,25 form actually stimulates bone deposition. The effect of increasing production of 24,25-dihydroxyvitamin D₃ while decreasing production of 1,25-dihydroxyvitamin D₃ has a favorable effect. The ratio of 24,25 to 1,25 changes from 186 to 300 in captive denning bears (who have ample vitamin D in their summertime food rations) and from 16 to 89 in wild, denning bears.

The large increase in the ratio of 24,25 to 1,25 (61% in captive and 456% in wild bears) serves two purposes:

-   1. The ability of 1,25-dihydroxyvitamin D₃ to release calcium from     bone is reduced; and -   2. The increase in 24,25-dihydroxyvitamin D₃ is enough to recycle     calcium that continues to be lost from bone back into bone. The     ideal regulation of vitamin D metabolites to prevent high blood     calcium only works if the bear can prevent bone loss. We have found     that although the bears exists in a state similar to a     post-menopausal woman, the bear makes bone normally, protects its     skeleton from osteoporosis, and prevents high blood calcium and     death.

Female rats grow normally when receiving daily injections of BDI at a concentration similar to that which enters the blood stream each day from the urinary bladder of a denning bear. No untoward, observable signs or symptoms indicative of adverse reactions to BDI were observed in these rats.

Fasting Summer Bear Conclusions

The fasting summer bear exhibits substantially the same decrease in urea to creatinine ratio as the denning winter bear. Moreover, it exhibits essentially the same bone remodeling enhancement as the denning winter bear. Accordingly, the beneficial aspects of the bear isolate as it relates to renal disorders and osteoporosis appear to be equally as potent with the summer fasting bear as with the winter denning bear.

Evaluation of BDI and its Fractions

In Vivo Studies: Inducing Denning Bear Behavior in Guinea Pigs and In Vitro Studies: Stimulation of Bone Remodeling

In Vivo Studies

Introduction

The first study was exploratory. It evaluated BDI that had been isolated from winter urine. The second study determined the effects on vital signs of the guinea pig of a lyophilized sample of winter urine and of the precipitate isolated from the urine during deproteination. The third study used a Latin Square Design. It was an in-depth investigation of BDI and three of its isolated Fractions. The fourth study compared fifth study compared BDI derived from winter, denning bears with serum from active, eating bears. As described under “Chemistry of the Invention”, serum from winter, denning bears (BDI) and serum from active, eating bears were deproteinized with methanol, the proteins were pelleted by centrifugation, and the supernatants were removed and lyophilized. The dry samples were then reconstituted in 2 ml of saline.

Study One: Exploratory Study Comparing Effects of Summer and Winter Urine on Body Temperature. Heart Rates. and Tranquility in Guinea Pigs

Methods

Urine from denning and non-denning bears was processed in similar fashion. Guinea pigs received BDI in the same relative concentration as it appears in the denning bear. Thus, the predicted concentration in the blood of the guinea pig was about equal to the predicated concentration of BDI in the blood of the denning bear. Blood volume was estimated as five percent of body weight. 50 ml of urine was deproteinated, lyophilized, and reconstituted in 2 ml of sterile saline as described above. A 2 ml sample was delivered by intraperitoneal injection into each animal.

Results

Five minutes post injection, the animals receiving BDI presented signs of tranquility, reduced heart rate [from approximately 256 to 96 beats per minute (BPM)], and reduced body temperature (from approximately 38° C. to 35° C. or 100.4° F. to 95° F.). The tranquil effects lasted approximately 50 minutes. The tranquil effects were evidenced by the fact that animals could be held on their backs without signs of struggle and that the guinea pigs were alert to their surroundings, but were simultaneously very calm and indifferent to external stimuli such as sudden loud noises. Body temperatures did not return to normal for up to 15 to 20 hours post injection.

Guinea pigs receiving urine from non-denning bears that had been processed in a manner similar to the processing of BDI showed no decreases in body temperature or heart rate. They did not develop a tranquil state.

Conclusion

These data indicate that BDI induces responses of the denning bear in the guinea pig.

Study Two: Comparing Effects of Whole Urine and Precipitate on Heart Rates and Body Temperature in Guinea Pigs

Method

Four guinea pigs were injected with varying doses of lyophilized samples of winter bear urine or the precipitate resulting from deproteination of winter bear urine. Rectal body temperature was measured and an electrocardiogram (ECG) was taken every 15 minutes after time of injection. The material to be injected was prepared in the following manner.

Whole bear winter urine was aliquoted out into 20 ml, 40 ml, and two 50 ml samples.

The 20, 40, and one of the 50 ml samples were lyophilized and placed in the freezer until the day of the experiment.

The second 50 ml sample was treated with an equal volume of methanol, vortexed, and allowed to set in the freezer overnight.

The next day, the methanol treated urine was centrifuged and the supernatant removed.

The remaining precipitate was dried under a nitrogen stream and then frozen until the day of the experiment.

On the day of the experiment, each of the four samples were reconstituted into 2 ml of bacteriostatic 0.9% saline for injection. After a control ECG and rectal body temperature (° F.) were taken, each guinea pig was injected intraperitoneally. ECG recordings and rectal temperatures were then taken every 15 minutes for up to 90 minutes.

Results (Table 4 and Table 5)

The guinea pig receiving the protein precipitate (0.0148 g) had an average increase in heart rate of 18 bpm during the 90 minute observation period. The maximum change in heart rate was +28 bpm and occurred 15 minutes after injection. Rectal temperature changes ranged from −1.2° F. to +0.7° F.

The guinea pig that received the lyophilizate from 20 ml of urine (0.5384 g) exhibited an average decrease in heart rate of 49 bpm with the lowest heart rate measured at 15 minutes after injection. Rectal temperature decreased an average of 2.1° F. over the 90 minutes.

In the animal that received the lyophilizate from 40 ml of urine (1.2164 g), heart rate decreased by an average of 60 bpm within 15 minutes after injection. However, heart rate returned to normal more rapidly in this particular animal than in the guinea pig that received only 20 ml of the lyophilized urine. Therefore, the average change in heart rate for this animal was only −4 bpm. In contrast, rectal temperature decreased by 5.5° F. and remained lowered even at 90 minutes.

The guinea pig that received the highest dose of the lyophilizate from 50 ml of urine exhibited a maximum decrease in heart rate (−154 bpm) at 15 minutes. Rectal temperature decreased by 7.3° F. and was still 6° lower than control 90 minutes after injection.

All animals survived.

TABLE 4 GUINEA PIG STUDY: WHOLE URINE AND PRECIPITATE MEAN CHANGES IN HEART RATES (BPM) (Treated Rates - Control Rates) Post Injection Protein Time Precipitate 20 ml 40 ml 50 ml 15 minutes +28 −83 −60 −154 30 minutes +18 −34 +19 −129 50 minutes +17 −50 +15 −103 75 minutes +20 −43 −6 −135 90 minutes +9 −37 0 −120 Mean of Means +18.4 −49.4 −4.0 −128.2

TABLE 4 GUINEA PIG STUDY: WHOLE URINE AND PRECIPITATE CHANGES IN BODY TEMPERATURE (° F.) (Treatment Temperature - Control Temperature) Post Injection Protein Time Precipitate 20 ml 40 ml 50 ml 15 minutes — −0.5 −0.3 −4.3 30 minutes 0.0 −2.6 −2.8 −5.0 45 minutes +0.7 −4.4 −5.5 −7.3 60 minutes −1.2 −3.2 −5.3 −6.8 90 minutes −0.7 −0.0 −5.1 −6.8 Mean −0.3 −2.14 −3.8 −6.0 Summary

Fifty ml of winter bear urine that had been lyophilized and reconstituted in 2 ml of normal saline caused a 45% decrease in heart rate within 15 minutes of injection.

Fifty ml of winter bear urine that had been lyophilized and reconstituted in 2 ml of normal saline caused a decrease in rectal temperature that was maximal at 45 minutes post injection.

Both effects were sustained throughout the 90 minute observation period.

In the guinea pigs that received the lower doses of the lyophilizate from bear urine, heart rate and rectal temperature still decreased with maximal effects measured at 15 minutes for heart rate and 45 minutes for temperature.

The magnitude of the effects produced by 20 ml and 40 ml of urine were smaller when compared to 50 ml of urine.

The animal that received the precipitate intraperitoneally exhibited an increase in heart rate rather than a decrease with little or no change in rectal body temperature.

Conclusions

The lyophilized winter bear urine injected intraperitoneally into conscious guinea pigs produced a decrease in heart rate and rectal body temperature similar to changes previously noted with BDI. The precipitate from the same volume of urine did not produce the same effects; it did not decrease heart rate and had little or no effect on rectal body temperature.

Study Three: Latin Square Designed Studies—The Effect of BDI In A Non-Hibernating Animal. The Guinea Pig

Introduction

This study was designed to test the effects of BDI and its Fractions in guinea pigs. To ensure unbiased observations, the study was blinded so that the researchers did not know which animal was injected with BDI, with Fraction I, with Fraction II, with Fraction III, or with saline. The Latin Square Design permitted use of animals as their own controls. Thus, in each animal, changes in heart rate and temperature after experimental injections were compared to the guinea pig's own recorded normal heart rate and temperature prior to each injection. In addition, all animals received a control injection of sterile saline during the five week experimental period in an effort to measure the physiologic response in each animal to the pain of the injection itself. Food and water intake, urine output, and urea and creatinine excretion in urine were measured daily for four days after each injection. Therefore, each animal is used as its own control, and each sample injection can be compared to a saline control injection in all animals.

Methods

Heart rates were intermittently monitored by electrocardiograms. Rectal temperatures were intermittently monitored via inserted thermistors calibrated to National Bureau of Standard requirements. Recordings were made every 15 to 30 minutes throughout the two to three hour study. A video camera was used to record behavioral activity in each animal throughout the study. Research observers were asked to comment on each animals' tranquility by observing animal handling and animal reaction when exposed to a loud snapping noise. Thereafter, the animals were housed in a metabolic cage throughout the five-week experiment in order to measure food and water intake and urine output. Urine urea and creatinine concentrations were measured. Effects of the following fractions were compared with BDI, with the saline control, and with each other: Fraction I, representing BDI-[BHB+MNC]; Fraction II, representing BHB; and Fraction III, containing MNC.

Design

Fractions were obtained by combining appropriate samples from the second CCC run. They were lyophilized as those for BDI. Thereafter, they were reconstituted in a saline solution.

After collecting Fraction I, Fraction II, and Fraction III, the study was blinded so that the researchers did not know which animal was injected with Fraction I, with Fraction II, with Fraction III, with saline, or with BDI. Animals were used as their own controls in a Latin Square Design. Heart rates were intermittently monitored by electrocardiograms. Rectal temperatures were intermittently monitored via inserted thermistors. Results were recorded every 15 to 30 minutes throughout the two to three hour study. A video camera was used to record behavioral activity in each animal throughout the study. To measure effects on body temperature (° C.), heart rates (BPM), and tranquility from each injection on the five guinea pigs, the data were grouped into the following time categories: Zero minutes (pre-injection control), 15-25 minutes, 30-40 minutes, 41-59 minutes, 60-74 minutes, and 75-95 minutes (post injection). Each animal was used as its own control. Treatment means were reported as the difference of each injection effect from the zero minutes (control) result. Therefore, positive or negative treatment mean values indicate an increase or decrease in the effect measured. A similar approach was used for daily determinations of food and water intake and urine excretion of urea and creatinine.

Results

Body Temperature (Table 6)

Beginning at 30 minutes and extending through to the end of the study, BDI produced a significant reduction in body temperature. The overall mean of temperature reduction was seven fold greater than that experienced by the animal when it received saline as a control measure.

Effects of Fraction I, Fraction II, and Fraction III were not different from control observations throughout the study.

TABLE 6 GUINEA PIG STUDY: 5 × 5 LATIN SQUARE MEAN CHANGES IN BODY TEMPERATURE (° C.) Treatment Temperature − Control Temperature) Post Injection Time I II III BDI C P < 0.05 15 to 25 minutes 0.33 0.41 0.35 0.34 0.01 N.S. 30 to 40 minutes 0.10 0.34 0.19 −0.31 −0.31 N.S. 41 to 59 minutes 0.03 0.22 0.17 −0.84 −0.24 N.S. 60 to 74 minutes −0.15 0.21 0.10 −1.14 0.01 * 75 to 95 minutes −0.42 0.12 0.38 −1.54 −0.15 * Mean of Means −0.02 0.26 0.24 −0.70 −0.10 — I = BDI − (BHB + MNC) II = BHB III = MNC through Wash C = Saline Control *Treatments are significantly different at p < 0.05

BDI produced a significant reduction in heart rate. Animals receiving Fraction I showed a significant heart rate reduction of approximately 50% of that shown by BDI. Animals receiving Fraction III showed a moderate but not a statistically significant reduction in heart rate (approximately 20% of that shown by BDI). Compared to BDI, those receiving Fraction II showed only a 10% reduction in heart rate. Saline injection failed to reduce heart rate (Table 7).

TABLE 7 GUINEA PIG STUDY: 5 × 5 LATIN SQUARE MEAN CHANGES IN HEART RATES (Beats per Minute) (Treatment Rates − Control Rates) Post Injection Time I II III BDI C P < 0.05 15 to 25 minutes −34.4 −7.2 −15.2 −54.0 9.2 * 30 to 40 minutes −29.4 −4.4 −9.2 −53.0 6.8 ** 41 to 59 minutes −25.0 −7.6 −11.4 −62.8 6.8 * 60 to 74 minutes −19.8 2.2 −13.4 −53.8 4.4 N.S. 75 to 95 minutes −23.4 −7.6 −10.2 −51.6 0.2 N.S. Mean of Means −26.4 −4.9 −11.9 −55.0 5.5 — I = BDI − (BHB + MNC) II = BHB III = MNC through Wash C = Saline Control *Treatments are significantly different at p < 0.05 **Treatments are significantly different of p < 0.01 Food and Water Intake

Guinea pigs that received BDI showed a decreased intake of food that was significant by the third and fourth day post injection.

Water intake by guinea pigs that received BDI was not changed.

Urine urea to creatinine ratios were profoundly reduced in guinea pigs receiving BDI.

Tranquility (Table 8)

Only animals receiving BDI were rated more tranquil than those receiving saline.

TABLE 8 GUINEA PIG STUDY: 5 × 5 LATIN SQUARE TRANQUILITY Substance Fraction Number of Animals Tranquility* BDI — 5 3.6 BDI − (BHB + MNC) I 5 2.0 BHB II 5 2.8 MNC III 5 2.8 Saline (Control) C 5 2.6 *Animals rated 1 to 4 (anxious to tranquil) when exposed to a brief snapping sound and turned over on their backs. Death

Two animals died within 24 hours. One received Fraction I; the other received BDI.

Summary

BDI demonstrated significant and profound reductions in body temperature when compared to its Fractions—I, II, or III.

The reductions in body temperature stimulated by BDI increased over time with temperatures remaining low for up to 24 hours.

Individual components of BDI (Fraction I, Fraction II, and Fraction III) had no effect on body temperature.

BDI demonstrated significant and profound reductions in heart rate when compared to its Fractions—I, II, or III.

Heart rates were reduced significantly within 30 to 60 minutes after the injection of BDI and tended to return to normal within two to three hours post injection.

In order of responses, Fraction I, Fraction III, and Fraction II reduced, but to a much lesser degree, heart rates independently.

The decrease in urea to creatinine ratios were profoundly reduced in guinea pigs receiving BDI.

Only BDI induced tranquility over that shown by animals receiving the saline control.

Conclusion

BDI contains components that target specific physiologic changes independently, but BDI exhibits the greatest overall effects when all the components of BDI are present. The performance of BDI exceeds the results of any of the above fractional components.

Study Four: Effects of Combination of Fraction I. Fraction II. and Fraction III Isolated

From Urine in a Non-Hibernating Animal, the Guinea Pig

Introduction

Samples were defined as follows:

-   1. Combination A: Fraction I plus Fraction III representing BDI−BHB;     contains MNC. -   2. Combination B: Fraction I plus Fraction II representing BDI−MNC;     contains BHB. -   3. Combination C: Fraction II plus Fraction III representing     BHB+MNC.

The above Combinations were obtained by combining appropriate samples from the second CCC run. They were dried as those for BDI. Thereafter, they were reconstituted in a saline solution.

Methods

BDI obtained from urine taken from early, mid, and late denning bears was used for isolation of Fraction I, Fraction II, and Fraction III. The combinations were injected intraperitoneally.

Body temperature (° C.), heart rates (BPM), and tranquility were measured for each treatment on three guinea pigs.

Data were grouped into time categories: 0 minutes (pre-injection control), 30 minutes, 60 minutes, 75 minutes, and 260 minutes (post injection).

Each animal was used as its own control. Treatment means are reported as the difference of each treatment effect from the 0 minutes (control) result. Therefore, as in the Latin Square Study, positive or negative treatment mean values indicate an increase or decrease in the effect measured. A mean of the Combination means was then calculated from each Combination over all animals and all time categories. All research observers (blinded study) were asked to comment on each animals' tranquility by observing the animal handling and animal reaction when exposed to a loud snapping noise.

In these studies, comparison between guinea pigs, sample potency was expressed as the ratio of averaged treatment means to g dry weight of each sample injected.

Results

Temperatures (Table 9) were reduced in all three guinea pigs receiving Combination A, Combination B, and Combination C with the largest decreases in temperatures occurring in animals receiving Combination A or Combination B.

When temperature responses were related to weight of the injected sample (Table 9—Potency), Combination A, Combination B, and Combination C were potent in reducing body temperatures. Combination C had the greatest potency (Table 9).

TABLE 9 GUINEA PIG STUDY: COMBINED FRACTIONS CHANGES TN BODY TEMPERATURE (° C.) AND POTENCY (Treatment Temperature - Control Temperature) Post Injection Time Combination A Combination B Combination C 30 minutes −0.21 −0.67 −0.17 60 minutes −1.21 −1.68 −0.17 75 minutes −1.60 −2.01 −0.34 260 minutes  −4.49 −3.63 −1.50 Mean −1.88 −2.00 −0.55 Sample Weight 3.3833 g 1.9917 g 0.1699 g Potency* −0.56 −1.00 −3.24 Combination A = Fraction I + Fraction III = BDI − BHB (Contains MNC) Combination B = Fraction I + Fraction II = BDI − MNC (Contains BHB) Combination C = Fraction II + Fraction III = MNC + BHB (Through Wash)

Heart rates were reduced in all three guinea pigs. The largest reductions occurred in animals receiving combinations A and B (Table 10).

Combination C was most potent in reducing heart rate (Table 10).

TABLE 10 GUINEA PIG STUDY: COMBINED FRACTIONS MEAN CHANGES IN HEART RATES (Beats per Minute) AND POTENCY (Treatment Rates - Control Rates) Post Injection Time Combination A Combination B Combination C 30 minutes −88.0 −54.0 −14.0 60 minutes −70.0 −67.0 −50.0 75 minutes −79.0 −60.0 −68.0 Mean of Means −70.0 −60.3 −44.0 Sample Weight 3.3833 g 1.9917 g 0.1699 g Potency* −23.4 −30.3 −258.8 Combination A = Fraction I + Fraction III = BDI − BHB (Contains MNC) Combination B = Fraction I + Fraction II = BDI − MNC (Contains BHB) Combination C = Fraction II + Fraction III = MNC + BHB (Through Wash)

Combination A, Combination B, and Combination C produced tranquility in the animals (Table 11).

TABLE 11 GUINEA PIG STUDY: EFFECT OF COMBINED FRACTIONS, TRANQUILITY Number of Substance Combination Animals Tranquility* BDI − BHB Combination A 1 4.0 (Contains MNC (Fraction I + Fraction III) BDI − MNC Combination B 1 4.0 (Contains BHB) (Fraction I + Fraction II) MNC + BHB Combination C (Fraction II + Fraction III) 1 3.0 *Animals rated (anxious to tranquil) when exposed to a brief snapping sound and turned over on their backs.

Animals receiving Combination A or Combination B died within 24 to 48 hours post injection.

Summary

Combination A, Combination B, and Combination C greatly reduced body temperature and heart rate.

Reductions in body temperature increased over time with temperatures remaining low for up to 24 to 48 hours.

Heart rates were reduced within 30 to 60 minutes after the injections and remained low throughout the 75 minutes that the animals were monitored. Combination C gave the largest potency effect in temperature and heart rate reduction. The animal survived. This suggests that the components of Combination C may be the predominantly active ingredients in BDI containing no toxic side effects.

Conclusions

BDI from urine and its combined components demonstrate dramatic decreases in body temperature and heart rate in non-denning guinea pigs.

BDI from urine and its combined components also produce alert tranquility in this non-denning animal model.

Study Five: Comparison of BDI Derived from Denning Serum and Serum from Active Bears in a Non-Hibernating Animal the Guinea Pig

Methods

As previously described, equal volumes of BDI and summer active serum were processed by deproteinization, centrifugation, supernatant removed, lyophilization, and residue reconstitution into 2 ml of saline. The reconstituted samples were each intraperitoneally injected into guinea pigs. Body temperatures, heart rates, and tranquility ratings were recorded as described in Study One, Study Two, and Study Three.

Results

The mean decrease in body temperature associated with BDI was −0.19° C. This is approximately two-fold greater than the −0.10° C. shown by serum from active bears and by saline controls in the Latin Square Design.

No significant change in heart rates occurred after injection. BDI was associated with an overall mean decrease of 8 beats/minute; active bear serum showed a mean decrease of 7 beats/minute.

Neither animal showed signs of tranquility.

Conclusions

BDI from serum showed only a mild response in lowering body temperature.

Active bear serum showed no response in lowering body temperature.

Neither BDI from serum nor active bear serum affected the heart rate or induced tranquility.

The lack of response may be attributable to an extremely low concentration of BDI in the samples.

Overall Conclusions of Guinea Pig Testing

When given intraperitoneally to the guinea pig, BDI induces the responses of the bear: tranquility, decreased heart rate, and decreased body temperature.

No differences in guinea pig results were noted when BDI was isolated from early, mid, or late denning bears.

BDI was most effective when used in full strength.

Isolated Fractions of BDI by themselves were inactive.

Combination of BDI into Combination A (Fraction I plus Fraction II), Combination B (Fraction I plus Fraction III), and Combination C (Fraction II plus Fraction III) also elicited positive results. Combination A and Combination B were associated with side effects which were, most likely, due to Fraction I. Three of seven animals died. They either received Fraction I or Combinations A and B that contained Fraction I.

A definite, safe, and highly active response with no observable side effects was noticed in the animal receiving purified Combination C (Fraction II plus Fraction III).

Treatment of Osteoporosis in Ovariectomized Rats

Our next step was to treat a living animal model similar to the post menopausal woman with BDI.

We used a pharmaceutical industry accepted animal model. Growing rats, less than six months old, were randomized into three groups of six rats each. One group was control (sham operated), one was ovariectomized, and one was ovariectomized and received BDI via subcutaneous injection. Similar volumes of saline were injected into the other two groups. BDI was given in amounts similar to its daily production in bears but proportionally scaled to body weight of the rat.

At the end of eight weeks, the ovariectomized group had become osteoporotic. When compared with this group, the ovariectomized group treated with BDI showed a 3% increase in bone mineral density (BMD) of the femur and a 4% increase in the lumbar vertebrae.

When compared with two month results of treating post menopausal women with estrogen, progesterone, and calcium, BDI results in rats showed a 16-fold greater increase in the BMD in lumbar vertebrae and a 3-fold greater increase in BMD of the femur. Another group of women on similar hormone replacement therapy showed only a 1.7% increase in BMD of the lumbar spine even though they were treated for 1.6 years.

In Vitro Studies: Evaluation of BDI and its Fractions in Stimulating Bone Remodeling

Introduction

These studies focused on serum and urine obtained from denning bears. The bone mass of denning bears remains constant even though they exist in a non-weight bearing state, a condition that induces loss of bone. Unlike other mammals, the bear maintains bone mass, structure, and strength. In the bear, the cells that produce bone (osteoblasts) are as active as the cells that resorb bone (osteoclasts). Under similar conditions, other mammals (including humans) lose bone by reducing bone formation, by maintaining or increasing bone resorption, or by a combination of these changes.

Test One: Inhibition of the Resorption Activity of Chicken Osteoclasts

Introduction

Unprocessed serum from active eating bears and unprocessed serum from denning bears both showed an inhibition of osteoclast resorption activity. The studies focused on the denning bear because it continues to make bone despite the fact that its non-weight bearing state lasts for months.

Methods

BDI Serum Studies (Table 12)

BDI, BHB, and BDI−BHB (containing MNC) were prepared from serum of bears as described under “Chemistry of the Invention” in this application.

Results

BDI from three bears in concentrations of 1 mg/ml of sample reduced osteoclast resorption activity to values of 24, 46, and 55 percent of control. More dilute samples were not effective (0.1, 0.01, 0.001 mg/ml).

The sample BDI−BHB that contains MNC also proved effective in two bears at concentrations of 1 mg/ml, reducing osteoclast resorption activity to 10 and 75 percent of control.

BHB by itself had no effect on osteoclast resorption.

TABLE 12 BEAR SERUM: INHIBITION OF FORMATION OF CHICKEN OSTEOCLASTS FROM CHICKEN MONOCYTES OBTAINED FROM BONE MARROW Percent Reduction from Control Concentration of Bear Weight CCC Test Sample (mg/ml) Substance Name (g) Samples 0.001 0.01 0.1 1.0 BDI Amanzo 0.017 Not run 125 115 108 55 Caruso 0.012 Not run 80 106 46 UP 0.020 Not run 152 93 90 24 BDI − BHB Amanzo 0.026 Fraction 119 103 108 75 (Contains I and III MNC) UP 0.078 Fraction 84 90 60 10 I and III BHB Amanzo 0.0006 Fraction 130 130 135 II Caruso 0.0023 Fraction 95 95 II UP 0.002 Fraction 80 105 110 II Conclusion

Direct action of BDI isolated from serum with or without BHB produced an environment conducive for bone formation by inhibiting resorption activity of osteoclasts, the cells that dissolve bone.

BDI Urine Studies (Table 13)

Methods

BDI was prepared from urine from three bears as described previously under “Chemistry of the Invention” of this application.

Results

BDI in concentrations of 10 mg/ml of sample inhibited resorption activity of osteoclasts to values of 25, 35, and 38 percent of control. More dilute samples were not effective (Table 13).

TABLE 13 BEAR URINE: INHIBITION OF FORMATION OF CHICKEN OSTEOCLASTS FROM CHICKEN MONOCYTES OBTAINED FROM BONE MARROW Percent Reduction from Control Bear Weight Concentration of Test Sample (mg/ml) Substance Name (g) 0.01 0.1 1 3 10 BDI Amanzo 0.268 147 110 130 95 25 Caruso 0.255 125  85 35 UP 0.270 123 107 38 Conclusions

BDI isolated from urine induces bone formation by inhibiting bone resorption by osteoclasts.

BDI isolated from serum is approximately 10 times more effective than BDI isolated from urine in reducing bone resorption by osteoclasts.

Test Two: Simultaneous Evaluation of Osteoblast and Osteoclast Activity

Methods and Materials

Experiments utilized an in vitro bone culture system. Calvaria (skull) of 4 to 6 day old neonatal mice were dissected out and cultured in individual capped test tubes in 2 ml of culture media (DMEM+glutamine, heparin, inactivated horse serum, and antibiotics). Each calvaria was gassed and incubated in a rotating roller drum at 37° C. Osteoblast activation (increased bone formation) was evaluated as a function of alkaline phosphatase activity (ALP). Osteoclast activity (bone resorption) was evaluated as a function of beta-glucuronidase activity. For testing purposes, two samples of serum from bears were used: 1) unprocessed bear serum, and 2) processed bear serum (BDI). Horse serum was used as a serum control to ensure that stimulation was not due to serum growth factors.

Results

Unprocessed bear serum from active, eating, weight-bearing bears increased ALP activity from 600 to 1200 nmole ALP/bone/30 minutes.

Unprocessed bear serum from denning, non-eating, non-active, non-weight bearing bears also significantly increased ALP activity from 600 to 1200 nmole ALP/bone/30 minutes.

Horse serum showed no change in ALP activity.

Unprocessed bear serum from denning bears showed a dose response result. The saline control value of 250 ALP/bone/30 minutes significantly increased to 600, to 800, and to 1000 ALP/bone/30 minutes in response to 50, 100, and 200 μl of serum respectively.

BDI increased ALP activity from 310 to 520 ALP/bone/30 minutes, about 55% of the response elicited by unprocessed bear serum that, in the same test, increased ALP to 700 ALP/bone/30 minutes.

The ability of BDI to increase ALP activity proved significantly greater than effects of calcitonin.

Inactivating serum proteins in unprocessed bear serum by heat produced results similar to BDI; ALP activity increased.

BDI failed to activate beta-glucuronidase. Combining these findings with the above indicated that BDI primarily stimulated bone formation by osteoblasts.

Unprocessed serum from active and denning bears showed both mild stimulation and failure to stimulate beta glucuronidase activity. However, when osteoclasts were stimulated, the response was less than one-half of the osteoblast stimulatory response. Therefore, bone formation activity continued to exceed bone resorbing activity.

Conclusions

Unprocessed serum from active and denning bears stimulates osteoblasts.

Unprocessed serum from active and denning bears varied in its ability to stimulate osteoclasts. At times no changes were observed; at other times mild stimulation was observed.

BDI stimulates osteoblasts to about 55% of that shown by unprocessed serum.

BDI does not stimulate osteoclasts.

The overall effect on bone remodeling is creation of an environment conducive to bone formation—stimulation of the limb that forms bone (osteoblasts) while not stimulating bone resorption (osteoclasts).

Test Three: The Effect of Summer Fasted BDI on Osteoblast and Osteoclast Activities

Introduction

As previously described, fasted bears (who had access to water) during the summertime revealed changes in levels of serum urea, creatinine, and a U/C ratio similar to changes noted when bears were denning. Thus, it was concluded that the summer fasting bears were in the mode of urea recycling (See Tables 1 and 2). Test Three was done to determine if bone remodeling was also stimulated when the bears were fasting. The effect of the 21 day summer fast on bone remodeling was determined by evaluating the activity of BDI obtained from these bears in an in vitro bone culture system.

Materials and Methods

As described in the discussion Test Two, calvaria of 4 to 6 day old neonatal mice were used for the in vitro bone culture system. Alkaline phosphatase activity (ALP) was used as a means of evaluating osteoblast activity (increased bone formation).

Because previous tests using beta glucuronidase activity to evaluate osteoclast activity (increased bone resorption) were inconclusive, a more sensitive test was employed. The production of tartrate resistant acid phosphatase (TRAP) was used as a measure of osteoclast activity (Lau et al., 1987; Delamis 1988). For testing purposes, BDI was prepared from urine of bears before and at the end of the 21 day fast. Denning bear plasma served as a positive control. Pre-fasted BDI was compared with fasted BDI. Both were compared with denning bear plasma and all three samples were compared with the phosphate buffered saline control.

Results

Osteoblast Results (Table 14)

Pre-fasted BDI results were similar to results of denning bear plasma. Both showed a moderate, significant increase in osteoblast activity (55% and 50% above control respectively). However, BDI from the final day of fasting significantly stimulated osteoblasts some 300% above control, about a six-fold increase over results from denning bear plasma or pre-fasted BDI.

TABLE 14 Changes in Medium Alkaline Phosphatase Activity In Calvaria Incubated with Normal Denning Bear Plasma and BDI Processed from Urine Before and At the End of a 21-Day Fast Treatment Group ALP Activity¹ PBS (Phosphate Buffered Saline) 444.8* ± 108.5  BP (Denning Blood Plasma) 666.4^(a,b) ± 127.2   Fasted (BDI from Urine of Fasted 1337.7^(c) ± 346.3    Bears) Pre-Fasted (BDI from Urine of Non- 690.9^(b) ± 120.9  Fasting Bears) ¹mnol of p-nitrophenol/30 min/bone Different letters indicate a significant difference, p < 0.05, n = 6 Osteoclast Results (Table 15)

When using TRAP as an indicator of osteoclast activity, results clearly demonstrate BDI's ability to inhibit osteoclast function. Both the fasted and pre-fasted results showed similar, significant inhibitory effects on osteoclast function, reaching levels 40% to 46% of normal. These results confirmed results using the chicken osteoclast tissue culture assay (Tables 12 and 13) as an indicator of osteoclast activity. Denning bear plasma showed no effects on osteoclast function.

TABLE 15 Changes in Medium Tartate Resistant Acid Phosphatase Activity In Calvaria Incubated With Normal Denning Bear Serum and BDI Processed from Urine Before and at the End of a 21-Day Fast Treatment Group TRAP Activity² PBS (Phosphate Buffered Saline) 142.5^(a) ± 53.5 BP (Blood Plasma) 182.8^(a) ± 58.2 Fasted (BDI from Urine of Fasted  77.4^(b) ± 4.1 Bears) Pre-Fasted (BDI from Urine of Non-  84.0^(b) ± 4.9 Fasting Bears) ²mnol of p-nitrophenol/60 min/bone Different letters indicate a difference from the phosphate buffered saline control, p < 0.05, n = 6 Conclusions

Summer fasting in black bears induces a significant increase in potency of BDI in stimulating bone formation through activation of osteoblasts. Simultaneously, BDI significantly inhibits osteoclast activity. Thus, fasting in summer potentiates BDI's ability to stimulate bone formation.

Overall Conclusions of Bone Remodeling Studies

Results of the two separate studies independently performed at two institutions in two different states show complementary findings that support the conclusion that BDI stimulates bone formation and inhibits bone resorption since: BDI stimulates osteoblasts to form bone, BDI does not stimulate osteoclasts already present in bone, BDI inhibits resorption of bone by osteoclasts, and the net effect of these changes is to form bone. Summer fasting induces similar results in bone remodeling.

BDI is extremely potent since it stimulates the bone forming process while simultaneously inhibiting the bone resorption process of bone remodeling. Summer fasting in bears duplicates these positive findings found in denning bears.

Occurrence of Fraction II (BHB) and Fraction III (MNC) In Fasting. Adult Humans

Methods and Materials

Initially, BHB was identified by TLC/ninhydrin in very low concentrations in serum samples obtained from two humans that fasted for 20 hours. The serum samples were also deproteinated using the same method established for BDI. A follow-up study was done in fifty adult humans who had fasted for twenty hours to determine if components contained in BDI, namely BHB and MNC, could be found.

Results

MNC was not detected in the serum of fasting humans.

BHB appeared in serum samples obtained from subjects after a food restricted 20 hour fast.

BHB was not detected in serum samples obtained from subjects in the fed state.

Little to no BHB was detected in the urine of subjects collected before and after the 20 hour fast.

Conclusions

MNC, found in BDI, was not found in fasting human serum or urine.

Serum and urine from fasting humans contains BHB.

Dosage Formulations

After BDI (containing both BHB and MNC) alone or in combination with existing identified metabolites of denning bears which are also found in humans, has been isolated as set forth above, it is combined with desirable solvents such as saline or 5% dextrose in water.

After the solvents have been applied, a carrier may also be involved. Such carriers include: peanut oil, propylene glycol, a 5% alcohol based elixir, or pills and capsules containing lactose and/or calcium carbonate fillers. Transdermals are available as an alternative means of delivering the necessary doses of BDI. For subcutaneous, intramuscular, intravenous, or other specialized routes such as into the cerebral spinal fluid, appropriate carriers such as saline, Ringer's lactate, or dextrose solutions may be used. BDI is stable, water soluble, and will not suffer dissolution after stirring or settling overnight.

Once the syringe has been loaded, or the pill compounded, the maximum dosages (which must first be assessed for safety) are calculated for the animal to be tested. The present means to predict maximum dosage was based only on the lyophilized BDI contained in aliquots of 50 ml of denning bear urine that also contained 200 micrograms (μg) of MNC. Next, the blood volume of the recipient is equated with 50 ml urine volumes from the bear. The concentration of MNC in 50 ml of urine is used for calculations.

Mammals have blood volumes of approximately 5% of total body weight. Therefore, a 1000 gram guinea pig has 0.05×1000 g=50 ml blood.

Fifty milliliters of denning bear urine containing between 2.0 and 3.6 grams of BDI also contains 200 micrograms (μg) MNC or 4 μg/ml.

Therefore, the dosage and formulation for a 1000 gram guinea pig was BDI containing 200 μg MNC, which equaled a dose of 0.2 μg MNC/g body weight.

Reaffirmation of Findings: Urea Recycling is Produced when BDI Infected into Guinea Pigs but not Necessarily its Basic Components.

A urea creatinine ratio indicative of urea recycling (10 or less) was produced when BDI was injected into guinea pigs. This effect of efficient recycling lasted for three days after the injection. BDI was then separated into its three basic components. These were done previously as set forth in connection with the Table 1. The three basic components were BDI minus (BHB+MNC); BHB; and MNC. When each of these three basic components was injected separately into guinea pigs, the urine of guinea pigs did not exhibit a urea to creatinine ratio indicative of urea recycling (see Table 16).

TABLE 16 Urine Urea to Creatine Ratio in Guinea Pigs For Three Days Post-Injection Treatment Day 1 Day 2 Day 3 Control: Average U/C Ratio 34.28 34.28 34.28 Group A: BDI − (BHB + MNC) 26.33 22.13 26.09 (Contains 0.185 g urea) Group B: BHB 31.86 29.45 23.69 Group C: MNC Through Wash 26.23 33.20 34.55 Group D: BD1 (Contains 1.1 g urea) 8.33 12.25  7.66 I Group E: Saline Control 17.39 13.01 14.93

Thus, the combination of some substances contained in Fractions 1-17 of Table 1 (BDI minus [BHB+MNC]) and some substances from the fractions associated with BHB and/or MNC stimulate urea recycling.

Some of the individual components of these fractions are now known. The combination of the active substances in each fraction will stimulate urea recycling in the guinea pig, as distinguished from the lack of significant recycling when the three separate components are injected separately.

Further Refinement of Separation Techniques for BDI Isolated from Denning Bear Urine to: 1) Search for the Fractions in BDI Responsible for Stimulation of Osteoblasts. 2) Identify Known Chemicals in the Ten Fractions of BDI. and 3) Further Purify the Fractions of BDI by HPLC in Order to Identify Structural Components of MNC by Nuclear Magnetic Resonance and Mass Spectrometry.

Chemical methods of obtaining BDI fractions and isolating the same were performed as previously set forth in Table 1. To support further analysis, ten newly defined fractions from the countercurrent coil were collected. For example, the new Fraction I was obtained by pooling the first five elutions acquired from the countercurrent centrifuge. Total volume per collection tube was 20 ml; therefore, Fraction I contains 100 ml.

The precise countercurrent apparatus and centrifuge is manufactured by P.C., Inc. of Potomac, Md., referred to as a Multi-Layer Coil CCC. The #10 coil having a volume of 385 ml was used in processing all of the elutions and rinse which resulted in new Fractions I-X (Table 17).

TABLE 17 Separation of BDI Into Ten Fractions After CCC New Fractions CCC Fractions Fraction I 1-5 Fraction II  6-10 Fraction III 11-15 Fraction IV 16-20 Fraction V 21-25 Fraction VI 26-30 Fraction VII 31-35 Fraction VIII 36-40 Fraction IX 41-45 Fraction X Methanol Wash

The mobile phase (lower phase of 1-butanol:water:acetic acid, 20:20:1 mixture) of the first six of ten fractions were pumped through the CCC at 4 ml/minute. Collections were taken every twenty-five minutes. After collection of Fraction VI, the coil was stopped. Mobile phase continued pumping at an increased rate of 10 ml/minute. Collections were made at ten minute intervals. The mobile phase was discontinued while a 1:1 mixture of methanol and water was begun before beginning collection of Fraction IX. The methanol/water mixture was switched to 100% methanol at the beginning of Fraction X. After ten minutes, the pump was stopped and the coil was emptied by forcing compressed air through it. Everything collected from the coil at this point was added to Fraction X. All fractions were stored at −70° C. until lyophilization.

Search for Site of Osteoblast Stimulation in BDI

A sample of urine collected from a single denning bear was deproteinated and lyophilized. Up to one gram of BDI was then loaded on the CCC and separated into ten fractions through the procedure diagrammed in Table 17. Weights were obtained for each fraction. Fractions obtained from four separate runs of the CCC were combined before use in osteoblast cultures.

Each combined fraction was tested in a mouse calvaria bioassay to determine its effectiveness in stimulating osteoblasts. An increase in alkaline phosphatase production was interpreted as osteoblast stimulation.

The ability of each combined fraction to stimulate alkaline phosphatase in the mouse calvaria bioassay was measured and expressed as a percent of control. This was compared to the ability of BDI and of pooled blood serum from denning bears to stimulate alkaline phosphatase in the mouse calvaria bioassay (Table 18).

TABLE 18 Percent Stimulation of Osteoblast Activity By Blood Serum. Bear Derived Isolate. and Its Fractions Percent Above Control/mg Sample Specimen Fraction III 23 Fraction II 78 BDI (Bear Derived Isolate) 75 BS (Blood Serum) 322 Fraction X 292 Fraction IV 401 Fraction IX 571 Fraction V 3,740 Fraction VI 4,281 Fraction VII 37,432 Fraction II, BDI, Pooled blood serum from denning bears, Fraction X, Fraction IV, Fraction IX, Fraction V, Fraction VI, and Fraction VII demonstrated stimulation of osteoblast activity. Fraction III inhibited osteoblast activity. Thus, Fraction III has the potential to arrest Paget's disease and other forms of neoplasms such as cancer resulting from overactivity of osteoblastic-induced bone growth. For a list of substances identified for Fraction III see Tables 19 and 20.

TABLE 19 QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS FRACTION 111, BEAR URINE JZ4061: 5 mM/M CREATININE Nrml Range Organic Acids LACTIC ACID 0  0-75 PYRUVIC ACID 0  0-20 GLYCOLIC ACID 6  0-50 ALPHA-OH-BUTYRIC 0.0 0-1 OXALIC 0.0  0-25 4-OH-BUTYRIC 0.0 0-1 HEXANOIC ACID 0.2  0-11 5-HYDROXYCAPROIC 4.4 0-1 OCTANOIC 0.0 0-1 BETA-LACTATE 0.0 0-8 SUCCINIC ACID 0  0-20 GLUTARIC ACID 0.4 0-2 2-OXO-GLUTARATE 0  0-210 FUMARIC 0.0 0-5 MALEIC 0.0 0 MALIC ACID 28.1 0-2 ADIPIC ACID 0.0 0-7 SUBERIC ACID 1.0  0-11 SEBACIC ACID 0.0 0-2 GLYCERIC ACID 0 0-4 BETA-OH-BUTYRIC 0 0-3 METHYLSUCCINIC 0.0 0 METHYLMALONIC 0 0-5 ETHYLMALONIC 0.0 0-4 HOMOGENTISIC ACID 0.0 0-1 PHENYLPYRUVIC ACID 0.1 0-1 SUCCINYLACETONE 0.0 0-1 3-OH-ISOVALERIC 0.0  0-21 PHOSPHATE 90   0-3000 CITRIC ACID 24  0-450 HIPPURIC ACID 11   0-2000 URIC ACID 0  0-360 Nutritionals KYNURENIC ACID 0.6 FORMIMINOGLUTAMIC 0.15 0-3 4-PYRIDOXIC ACID 0.2 0-9 PANTOTHENIC ACID 14  0-30 XANTHURENIC ACID 0.0 0-1 KYNURENINE 0.1 0-1 QUINOLINIC 0.0 0-6 OROTIC ACID 0.00 0-3 D-AM LEVULINIC 4.0  0-18 3-METHYL HISTIDINE 0  0-75 NIACINAMIDE 0.0 0-1 PSEUDOURIDINE 58  10-220 2-DEOXYTETRONIC 0  0-75 P-HO-PHEN-ACETIC 0  0-12 XANTHINE 0  0-18 UROCANIC ACID 0 0-3 ABSCORBIC ACID 1  0-160 GLYCEROL 0 0-9 Carbohydrates THREITOL 0  0-40 ERYTHRITOL 0  0-55 ARABINOSE 0  0-30 FUCOSE 0.7  0-12 RIBOSE 3.2  0-12 XYLOSE 0  0-70 FRUCTOSE 0  0-115 GLUCOSE 3  0-110 GALACTOSE 20  0-200 MANNOSE 10  0-70 N-AC-GLUCOSAMINE 1.0 0-3 LACTOSE 2  0-60 MALTOSE 1  0-40 XYLITOL 0.1  0-15 ARABINITOL 0.0  0-30 RIBITOL 0.0  0-10 ALLOSE 1.4  0-10 GLUCURONIC ACID 113.6  0-50 GALACTONIC ACID 12  0-60 GLUCONIC ACID 5.2  0-35 GLUCARIC 2.2 0-5 MANNITOL 11.5  0-15 DULCITOL 2.2  0-10 SORBITOL 3.2  0-10 INOSITOL 3.4  0-12 SUCROSE 0  0-75 Neurotransmitters GABA 0.0 0-1 HOMOVANILLIC ACID 0.0  0-10 NORMETANEPHRINE 0.0 0-1 VANILLYLMANDELIC 0.0 0-6 METANEPHRINE 0.1 0-2 5-HIAA 0.0 0-6 MHPG 0.0 0-1 ETHANOLAMINE 0 10-90 Amino Acids and Glycine Conjugates PROPIONYL GLY 0.3 0-1 BUTYRYL GLYCINE 0.1 0-1 HEXANOYL GLYCINE 0.1 0-1 PHENYL PROP GLY 0.0 0-1 SUBERYL GLYCINE 0.0 0-1 ISOVALERYL GLY 0.0 0-1 TIGLY GLY 0.0 0-1 BETA MET CROT GLY 0.0 0-1 GLYCINE 1  0-500 ALANINE 2  0-130 SARCOSINE 0.0 0-8 BETA-ALANINE 0.1 0-2 B-AMINOISOBUTYRIC 0  0-50 SERINE 0  0-85 PROLINE 0.0 0-8 HYDROXY PROLINE 0  0-75 HYDROXY LYSINE 0.1 0-1 ASPARTIC ACID 0.0 0-2 ASPARAGINE 0.0 0-2 N-AC ASPARTIC 0.0  0-20 ORNITHINE 0.1 0-5 GLUTAMIC ACID 0.1 0-6 GLUTAMINE 1  0-210 PIPECOLIC ACID 0.1 0-1 LEUCINE 0.0 0-9 KETO LEUCINE 0.0 0-1 VALINE 0.0  0-18 KETO-VALINE 0.0 0-1 ISOLEUCINE 0.0 0-5 KETO-ISOLEUCINE 1.0 0-1 LYSINE 1  0-35 HISTIDINE 1  0-225 THREONINE 0  0-45 HOMOSERINE 0.3 0-1 METHIONINE 0.0 0-3 CYSTEINE 0  0-160 HOMOCYSTEINE 0.0 0-1 CYSTATHIONINE 0.1 0-1 HOMOCYSTINE 0.0 0-1 CYSTINE 0.1 0-5 PHENYLALANINE 16  0-20 TYROSINE 1  0-22 TRYPTOPHAN 0  0-25

TABLE 20 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS FRACTION III. BEAR URINE JZ4061 CONCENTRATION: THIS SAMPLE CONTAINED 20.72 mM CREATININE/mL LIB FIT AREA PEAK # CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY vs 1000 AREA % OF CREAT 18 24, NU3131 2125 767 1.18 72.24 25 25 0 0 2.75 167.69 32 32 0 0 0.07 4.42 57 57 0 0 0.14 8.41 68 1,3 PROPANEDIOL DI-TMS 1675 854 0.35 21.28 78 78 0 0 0.30 18.24 83 PROPENE GLYCOL DI-TMS 50 868 0.86 52.40 94 GLYCOLIC ACID DI-TMS 55 925 1.83 111.85 97 GLYCOLIC ACID DI-TMS 55 947 1.46 88.88 101 92, NA3011 2070 711 0.09 5.63 112 104, NJ3031 2131 834 1.87 114.25 181 107, KA1051 2050 712 0.08 4.73 243 4-HYDROXY BUTYRIC ACID DI-TMS 97 799 0.12 7.40 257 MALONIC ACID DI-TMS 100 760 0.09 5.38 323 PHOSPHATE TRI-TMS 1413 929 0.16 9.94 351 PHOSPHATE TRI-TMS 1413 834 0.13 7.80 357 PHOSPHATE TRI-TMS 1413 852 0.60 36.50 362 PHOSPHATE TRI-TMS 1413 925 0.41 25.17 382 PHOSPHATE TRI-TMS 1413 933 0.08 4.58 387 PHOSPHATE TRI-TMS 1413 804 0.70 42.71 409 409 0 0 0.23 14.03 423 409, JZ4061 2327 959 0.73 44.75 430 409, JZ4061 2327 928 0.58 35.39 462 283, NF3091 2093 733 0.12 7.05 486 GLYCERIC ACID TRI-TMS 324 626 0.75 45.99 513 283, NF3091 2093 747 0.11 6.47 527 283, NF3091 2093 745 0.18 11.14 600 2,4 DIHYDROXYBUTYRIC ACID TRI-TMS 1889 922 0.23 13.89 628 628 0 0 0.09 5.22 638 3,4 DIHYDROXY BUTYRIC ACID TRI-TMS 361 887 0.88 53.73 658 CITRAMALIC ACID TRI-TMS, 675 2103 703 0.13 8.17 664 645, M27041 1836 863 0.13 7.74 694 CITRAMALIC ACID TRI-TMS, 675 2103 940 0.13 10.30 738 2-DEOXY PENTONIC ACID GAMMA LACTONE DI-TMS 176 795 0.15 8.91 764 1-AMINO CYCLOPENTANE CARBOXYLIC ACID DI-TMS 158 614 4.40 268.70 773 TETROSE TRI-TMS 362 938 3.31 202.06 787 TETROSE TRI-TMS 362 941 9.36 571.10 800 3-METHYL-2-TENTENEDIOIC ACID DI-TMS 2004 726 0.07 4.32 813 CREATININE ENOL TRI-TMS 1467 865 1.68 102.41 819 TETROSE TRI-TMS 362 683 1.09 66.57 825 4 DE-O TETRONIC TMS3, THREO 1649 671 0.65 39.52 836 4 DE-O TETRONIC TMS3, THREO 1649 902 5.55 338.69 859 4 DE-O TETRONIC TMS3; THREO 1649 886 1.97 120.42 886 ALANINE DI-TMS 78 546 0.08 5.08 903 PARA HYDROXY BENZOIC DI-TMS 202 635 0.07 4.53 910 D-ERYTHRO-PENTITOL, 2-DEOXY-1,3,4,5-TETRAKIS- 633 742 0.31 18.65 927 2,2 DIMETHYL 3-HYDROXY BUTRIC ACID DI-TMS 180 546 0.58 35.27 943 LACTULOSE METABOLITE? 1751 847 0.76 46.27 951 ARABINOFURANOSE TETRA-TMS 675 855 0.26 16.12 963 GLYCOLIC ACID DI-TMS 55 319 0.97 59.40 972 981, M21021 1829 752 0.46 27.86 985 RIBULOSE PER-TMS 1848 749 0.88 53.83 996 996 0 0 1.31 79.71 1005 965, JJ4011 2191 708 0.27 16.69 1011 ARABITOL 1841 752 0.31 19.21 1019 ARABITOL 1841 664 0.15 9.44 1024 1024 0 0 0.30 18.25 1034 D-ERYTHRO-HEX-2-ENOUIC ACID, DI-O-METHYLBIS-O 404 581 0.07 4.18 1041 6-DEOXY MANNOSE TETRA-TMS 719 873 0.28 16.91 1054 ARABITOL 1841 959 2.43 148.36 1060 ARABINONIC ACID, 2,3,5-TRIS-O-TMS-, GAMMA-L 464 731 0.17 10.45 1072 ARABITOL 1841 951 4.16 254.05 1077 1073, RT1051 2040 732 2.02 123.07 1099 CYSTEINE TRI-TMS 363 295 1.13 68.83 1107 D-XYLOPYRANOSE TETRA-TMS 679 783 0.93 56.63 1119 1357, M22011 1834 739 2.21 134.78 1126 6-DEOXY GLUCIOL PENTA-TMS 858 913 1.24 75.79 1131 1107, NU3081 2122 683 1.62 99.10 1138 4 DE-O TETRONIC TMS3, THREO 1649 691 0.97 59.50 1142 1142 0 0 0.11 6.54 1160 PROPANOIC ACID, 3-BIS TMS-OXY PHOSPHINYL OX 756 696 0.14 8.75 1167 CREATININE TETRA-TMS 1438 603 0.87 52.90 1176 ISO CITRIC ACID TETRA-TMS 775 891 3.14 191.49 1185 D-ARABINO-HEXITOL, 2-DEOXY-1,3,4,5,6-PENTAKIS 856 584 0.45 27.57 1195 1195 0 0 0.13 7.81 1203 1357, M22011 1834 683 1.48 90.53 1226 1224, YE1011 1884 638 0.99 60.32 1234 1234 0 0 0.08 5.12 1246 1246 0 0 0.99 60.46 1254 GALACTOSE PENTA-TMS 878 707 0.57 34.80 1258 NEO-INOSITOL HEXA-TMS 972 835 1.15 70.49 1269 BENZOIC ACID, 5-METHOXY-2-TMS-OXY-TRIMETH 293 336 0.33 20.21 1276 GLUCONIC ACID, 2,3,5,6-TETRAKIS-O-TMS-LACTO 737 816 0.73 44.42 1288 3,4,5 TRIHYDROXY FURAN 2-ACETALDEHYDE TETRA-T 743 680 0.31 18.72 1301 GLUCITOL TRI-TMS 979 899 1.51 92.20 1308 GLUCITOL TRI-TMS 979 895 1.60 97.44 1312 DULCITOL 1840 926 0.78 47.33 1318 1315, YE1011 1885 837 0.55 33.52 1325 2-DEOXY ERYTHROPENTONIC ACID TETRA-TMS 687 446 0.59 36.15 1334 GALACTONIC ACID HEXA-TMS 988 888 3.31 201.84 1354 TALOSE PENTA-TMS 896 883 0.45 27.31 1369 GALACTONIC ACID HEXA-TMS 988 789 0.58 35.69 1377 GALACTARIC ACID HEXA-TMS 993 772 0.46 27.82 1384 GALACTONIC ACID HEXA-TMS 988 811 0.83 50.75 1391 2-DEOXY ERYTHROPENTONIC ACID TETRA-TMS 687 529 0.20 12.26 1395 SCYLLO-INOSITOL HEXA-TMS 969 799 1.35 82.37 1403 BETA, PHENYLPYRUVIC ACID DI-TMS 280 205 0.59 36.22 1424 ARABITOL 1841 584 1.31 79.85 1438 ARABITOL 1841 548 0.78 47.66 1443 MUCO-INOSITOL HEXA-TMS 974 802 0.98 59.86 1451 XYLULOSE TETRA-TMS 1771 658 0.17 10.36 1460 1460 0 0 0.08 4.63 1473 1473 0 0 0.06 3.85 1484 1484 0 0 0.07 4.16 1504 1504 0 0 0.07 4.18 1553 .BETA, -D-GALACTOFURANOSE, 1,2,3,5,6-PENTAKIS- 880 625 0.09 5.69 1561 1561 0 0 0.29 17.73 1591 1591 0 0 0.06 3.84 1596 PSEUDO URIDINE PENTA-TMS 1779 792 1.91 116.63 1615 D-RIBOFURANOSE TETRA-TMS 685 762 0.65 39.75 1658 1658 0 0 0.27 16.45 1704 D-XYLOPYRANOSE TETRA-TMS 679 650 0.08 4.71 1726 ARABINONIC ACID, 2,3,4-TRIS-O-TMS-, LACTONE, 461 629 0.08 5.11 1801 6-DEOXY MANNOSE TETRA-TMS 719 855 0.20 12.13 *The named compound matches the sample peak with a reliability given by “FIT”/1000

When results of this bioassay were expressed per mg of sample to represent potency of the sample, Fraction V, Fraction VI, and Fraction VII demonstrated the highest potency (Table 18). Fraction V exhibited a fifty-fold increase in potency when compared with BDI and a twelve-fold increase over the pooled denning bear serum. Similarly, Fraction VI exhibited a fifty-seven fold increase in potency when compared with BDI and a thirteen-fold increase over the pooled denning bear serum; Fraction VII exhibited a five hundred fold increase in potency when compared with BDI and a one hundred seventeen fold increase over pooled denning bear serum.

Identification of Known Substances in the Ten Fractions of BDI

The ten fractions of BDI collected from the CCC (including Fraction III above) were submitted to Dr. James Shoemaker, Director of the Metabolic Screening Laboratory and Assistant Professor of Biochemistry and Medicine in the College of Medicine, St. Louis University, St. Louis, Mo., for analysis by gas chromatography and mass spectrometry (GC/MS). The mass spectra of trimethylsilyl derivatives of the compounds in the CCC fractions were compared to a database of more than forty thousand chemicals.

Tables 21 and 22 depict data generated from Fraction V. Tables 23 and 24 depict data generated from Fraction VI; Tables 25 and 26 depict data generated from Fraction VII. Data on retention times are available for the substances depicted in Tables 19 through 38.

TABLE 21 QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS FRACTION V, BEAR URINE JZ4081:7 um/L* Nrml Range Organic Acids LACTIC ACID 55124 PYRUVIC ACID 10460 GLYCOLIC ACID 1123 ALPHA-OH-BUTYRIC 1274.5 OXALIC 0.0 4-OH-BUTYRIC 0.0 HEXANOIC ACID 0.0 5-HYDROXYCAPROIC 0.0 OCTANOIC 0.0 BETA-LACTATE 0.0 SUCCINIC ACID 23256 GLUTARIC ACID 0.0 2-OXO-GLUTARATE ***** FUMARIC 0.0 MALEIC 0.0 MALIC ACID 0.0 ADIPIC ACID 0.0 SUBERIC ACID 0.0 SEBACIC ACID 0.0 GLYCERIC ACID 0.0 BETA-OH-BUTYRIC 2026.0 METHYLSUCCINIC 0.0 METHYLMALONIC 0.0 ETHYLMALONIC 0.0 HOMOGENTISIC ACID 0.0 PHENYLPYRUVIC ACID 0.0 SUCCINYLACETONE 0.0 3-OH-ISOVALERIC 231.5 PHOSPHATE 2.19 mg/dL CITRIC ACID 2865 HIPPURIC ACID 486 URIC ACID 0.59 mg/dL Nutritionals FORMIMINOGLUTAMIC 0.00 4-PYRIDOXIC ACID 0.0 PANTOTHENIC ACID 0 XANTHURENIC ACID 0.0 KYNURENINE 0.0 QUINOLINIC 1871.0 OROTIC ACID 0.0 D-AM LEVULINIC ***** 3-METHYL HISTIDINE ***** NIACINAMIDE 1121.0 PSEUDOURIDINE 11063 2-DEOXYTETRONIC 0 P-HO-PHEN-ACETIC 30 XANTHINE 0 UROCANIC ACID 0 ABSCORBIC ACID 0 GLYCEROL 7963.0 Carbohydrates THREITOL 0 ERYTHRITOL 0 ARABINOSE 0 FUCOSE 0.0 RIBOSE 0.0 XYLOSE 0 FRUCTOSE 0 GLUCOSE 23 mg/dL GALACTOSE 0 MANNOSE 84 N-AC-GLUCOSAMINE 0.0 LACTOSE 2869 MALTOSE 3113 XYLITOL 0.0 ARABINITOL 0.0 RIBITOL 0.0 ALLOSE 105.0 GLUCURONIC ACID 2467.5 GALACTONIC ACID 0 GLUCONIC ACID 0.0 GLUCARIC 0.0 MANNITOL 69.5 DULCITOL 0.0 SORBITOL 0.0 INOSITOL 0.0 SUCROSE 6311 Neurotransmitters GABA 562.0 HOMOVANILLIC ACID 0.0 NORMETANEPHRINE 0.0 VANILLYLMANDELIC ***** METANEPHRINE 20.0 5-HIAA 0.0 MHPG 500.0 ETHANOLAMINE 8655 Amino Acids and Glycine Conjugates PROPIONYL GLY 863.0 BUTYRYL GLYCINE ***** HEXANOYL GLYCINE 856.5 PHENYL PROP GLY 0.0 SUBERYL GLYCINE 49.0 ISOVALERYL GLY 0.0 TIGLY GLY ***** BETA MET CROT GLY 0.0 GLYCINE 15925 ALANINE 192 SARCOSINE 86.0 BETA-ALANINE 0.0 B-AMINOISOBUTYRIC 798 SERINE 12428 PROLINE 1351.0 HYDROXY PROLINE 15079 HYDROXY LYSINE 0.0 ASPARTIC ACID 3027.5 ASPARAGINE 0.0 N-AC ASPARTIC 0.0 ORNITHINE 393.5 GLUTAMIC ACID 952.5 GLUTAMINE 577 PIPECOLIC ACID 0.0 LEUCINE 1799.0 KETO LEUCINE ***** VALINE 3449.0 KETO-VALINE 0.0 ISOLEUCINE 1277.5 KETO-ISOLEUCINE 0.0 LYSINE 43 HISTIDINE 0 THREONINE 1750 HOMOSERINE 0.0 METHIONINE 599.0 CYSTEINE ***** HOMOCYSTEINE 0.0 CYSTATHIONINE 0.0 HOMOCYSTINE 0.0 CYSTINE 0.0 PHENYLALANINE 860.5 TYROSINE 1398 TRYPTOPHAN 183.5 THIS SAMPLE CONTAINED 130.58 mg Creatinine/dL *The numbers above are best used to make the qualitative judgement of normal versus abnormal and not for direct quantitative comparisons.

TABLE 22 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUTENTS FRACTION V. BEAR URINE JZ4081 CONCENTRATION: THIS SAMPLE CONTAINED 0.01 mM CREATININE/mL CREAT LIB FIT NOT PEAK # CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY vs 1000 AREA % FOUND 7 10, STN031 1893 783 4.08 19 16, 0I1031 1989 806 6.95 34 31, NF3031 2090 757 0.78 57 49, AK2011 2047 836 0.69 66 SILANE, TRIMETHYLPHENOXY- 1122 887 2.82 70 ETHYL AMINE DI-TMS 22 589 12.54 77 PROPENE GLYCOL DI-TMS 50 867 0.84 107 107, JZ4011 2301 787 0.79 117 104, NJ3031 2131 860 12.78 121 119, J04011 2243 922 1.09 185 BETA-LACTATE DI-TMS 1654 773 2.17 292 283, NF3091 2093 747 5.88 361 TRIMETHYLSILYL ETHER OF GLYCEROL 273 917 0.77 600 2-METHYL PROPANOATE GLYCINE CONJUGATE DI-TMS 226 904 0.88 707 BUTYRIC ACID GLYCINE CONJUGATE DI-TMS 225 904 2.12 805 METHYL D3 CREATININE TRI-TMS 1466 745 8.61 825 BUTANEDIOIC ACID, OXO-TMS-, BIS-TMS-ESTER 401 698 0.68 878 878 0 0 1.72 940 940 0 0 0.80 1076 CIS-ACONITIC ACID TRI-TMS 540 874 2.34 1111 SALICYLIC ACID DI-TMS ORTHO-HYDROXY-BENZOIC 1720 286 3.95 1135 1135, JZ4011 2306 865 1.88 1223 VANILLYL MANDELIC ACID TRI-TMS 610 898 1.73 1284 1284 0 0 1.01 1364 1364, JZ4011 2312 888 1.05 1594 1594 0 0 17.08 1604 FROM GUAIFENESIN, 1813, NH13041 2169 688 6.27 1788 1527, 0G1021 1987 631 1.79 *The named compound matches the sample peak with a reliability given by “FIT”/1000

TABLE 23 QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS FRACTION VI, BEAR URINE JZ4011: 1 mM/M CREATININE Nrml Range Organic Acids LACTIC ACID 2531  0-75 PYRUVIC ACID 516  0-20 GLYCOLIC ACID 53  0-50 ALPHA-OH-BUTYRIC 6.9  0-51 OXALIC 70.3  0-25 4-OH-BUTYRIC 0.0 0-1 HEXANOIC ACID 14.9  0-11 5-HYDROXYCAPROIC 0.0 0-1 OCTANOIC 0.0 0-1 BETA-LACTATE 29.4 0-8 SUCCINIC ACID 49  0-20 GLUTARIC ACID 272.8 0-2 2-OXO-GLUTARATE 26936  0-210 FUMARIC 24.1 0-5 MALEIC 0.0 0 MALIC ACID 1.5 0-2 ADIPIC ACID 3.7 0-7 SUBERIC ACID 5.7  0-11 SEBACIC ACID 0.0 0-2 GLYCERIC ACID 0 0-4 BETA-OH-BUTYRIC 55 0-3 METHYLSUCCINIC 8443.4 0 METHYLMALONIC 0 0-5 ETHYLMALONIC 0.0 0-4 HOMOGENTISIC ACID 25.6 0-1 PHENYLPYRUVIC ACID 7.7 0-1 SUCCINYLACETONE 2.6 0-1 3-OH-ISOVALERIC 0.6  0-21 PHOSPHATE 8   0-3000 CITRIC ACID 507  0-450 HIPPURIC ACID 472   0-2000 URIC ACID 218  0-360 Nutritionals KYNURENIC ACID 44.8 FORMIMINOGLUTAMIC 0.00 0-3 4-PYRIDOXIC ACID 0.0 0-9 PANTOTHENIC ACID 0  0-30 XANTHURENIC ACID 0.0 0-1 KYNURENINE 0.0 0-1 QUINOLINIC 0.0 0-6 OROTIC ACID 0.00 0-3 D-AM LEVALINIC 1657.1  0-18 3-METHYL HISTIDINE 2  0-75 NIACINAMIDE 16.3 0-1 PSEUDOURIDINE 12665  10-220 2-DEOXYTETRONIC 0  0-75 P-OH-PHEN-ACETIC 5  0-12 XANTHINE 38  0-18 UROCANIC ACID 47 0-3 ASCORBIC ACID 0  0-160 GLYCEROL 705 0-9 Carbohydrates THREITOL 0  0-40 ERYTHRITOL 12  0-55 ARABINOSE 0  0-30 FUCOSE 0.4  0-12 RIBOSE 0.7  0-12 XYLOSE 0  0-70 FRUCTOSE 135  0-115 GLUCOSE 99  0-110 GALACTOSE 12  0-200 MANNOSE 54  0-70 N-AC-GLUCOSAMINE 2.7 0-3 LACTOSE 259  0-60 MALTOSE 127  0-40 XYLITOL 0.0  0-15 ARABINITOL 0.0  0-30 RIBITOL 0.0  0-10 ALLOSE 0.3  0-10 GLUCURONIC ACID 10.2  0-50 GALACTONIC ACID 15  0-60 GLUCONIC ACID 1.0  0-35 GLUCARIC 0.2 0-5 MANNITOL 10.2  0-15 DULCITOL 0.4  0-10 SORBITOL 9.7  0-10 INOSITOL 8.5  0-12 SUCROSE 1349  0-75 Neurotransmitters GABA 1.0 0-1 HOMOVANILLIC ACID 5.6  0-10 NORMETANEPHRINE 41.3 0-1 VANILLYLMANDELIC 90.3 0-6 METANEPHRINE 1.1 0-2 5-HIAA 1.2 0-6 MHPG 0.0 0-1 ETHANOLAMINE 409 10-90 Amino Acids and Glycine Conjugates PROPIONYL GLY 0.0 0-1 BUTYRYL GLYCINE 1196.9 0-1 HEXANOYL GLYCINE 0.0 0-1 PHENYL PROP GLY 0.0 0-1 SUBERYL GLYCINE 0.0 0-1 ISOVALERYL GLY 0.0 0-1 TIGLY GLY 0.0 0-1 BETA MET CROT GLY 0.0 0-1 GLYCINE 1053  0-500 ALANINE 12  0-130 SARCOSINE 12.6 0-8 BETA-ALANINE 0.0 0-2 B-AMINOISOBUTYRIC 7  0-50 SERINE 1106  0-85 PROLINE 115.7 0-8 HYDROXY PROLINE 956  0-75 HYDROXY LYSINE 0.0 0-1 ASPARTIC ACID 232.4 0-2 ASPARAGINE 5.0 0-2 N-AC ASPARTIC 191.8  0-20 ORNITHINE 86.9 0-5 GLUTAMIC ACID 79.7 0-6 GLUTAMINE 4  0-210 PIPECOLIC ACID 0.0 0-1 LEUCINE 141.2 0-9 KETO LEUCINE 611.7 0-1 VALINE 272.9  0-18 KETO-VALINE 0.0 0-1 ISOLEUCINE 107.1 0-5 KETO-ISOLEUCINE 0.0 0-1 LYSINE 644  0-35 HISTIDINE 140  0-225 THREONINE 215  0-45 HOMOSERINE 0.0 0-1 METHIONINE 2.7 0-3 CYSTEINE 1122  0-160 HOMOCYSTEINE 0.0 0-1 CYSTATHIONINE 0.0 0-1 HOMOCYSTINE 0.0 0-1 CYSTINE 8.7 0-5 PHENYLALANINE 85  0-20 TYROSINE 68  0-22 TRYPTOPHAN 54  0-25 This sample contained 0.02 uMoles Creatinine/1.00 ml.

TABLE 24 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS FRACTION VI, BEAR URINE JZ4011 CONCENTRATION: THIS SAMPLE CONTAINED 0.02 uM CREATININE/ml LIB FIT AREA % PEAK # CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY VS 1000 AREA % OF CREAT 5 6, JI4081 2189 780 1.67 422.70 8 10, STN031 1893 857 2.71 684.47 20 16, 0I1031 1989 820 5.76 1454.73 35 35 0 0 0.75 190.42 58 49, AK2011 2047 835 0.52 132.24 67 SILANE, TRIMETHYLPHENOXY- 1122 932 2.18 551.58 73 1,3 PROPANEDIOL DI-TMS 1675 934 5.38 1358.88 78 LACTIC ACID DI-TMS 1510 927 0.74 187.43 107 107 0 0 0.59 148.59 118 104, NJ3031 2131 884 8.05 2032.64 122 119, JI4011 2243 925 0.82 206.86 134 BLYCINE DI-TMS 51 822 0.25 64.34 186 BETA-LACTATE DI-TMS 1654 755 1.55 391.09 251 251 0 0 0.38 95.36 294 UREA DI-TMS 37 800 3.00 757.29 362 TRIMETHYLSILYL ETHER OF GLYCEROL 273 904 1.33 336.55 383 OCTANOIC ACID, 2-0S0-, TRIMETHYLSILYL ESTER 72 707 0.27 69.11 427 METHYLSUCCINIC ACID DI-TMS 173 948 3.17 799.71 502 SERINE TRI-TMS 322 958 0.51 128.24 697 3-METHYL-2-PENTENEDIOIC ACID DI-TMS 2004 619 0.31 77.45 706 BUTYRIC ACID GLYCINE CONJUGATE DI-TMS 225 874 0.43 107.51 748 HYDROXY PROLINE DI-TMS 156 938 0.39 99.20 808 METHYL D3 CREATININE TRI-TMS 1466 705 12.91 3258.96 825 BUTANEDIOIC ACID, OXO-TMS-, BIS-TMS-ESTER 401 704 0.26 66.23 828 828 0 0 0.42 105.07 894 PENTANEDIOIC ACID, 3-OXO-, TRIS-TMS ESTER 448 923 0.46 116.34 901 PARA HYDROXY BENZOIC DI = TMS 202 912 0.38 95.59 964 964 0 0 1.16 293.82 1013 1013 0 0 0.39 97.24 1078 CIS-ACONITIC ACID TRI-TMS 540 839 6.15 1152.41 1111 P-HO PHENYL GLYCOLIC TRI-TMS 532 927 2.98 753.39 1135 1135 0 0 0.70 175.75 1141 1141 0 0 1.39 351.33 1167 CITRIC ACID TETRA-TMS 774 870 0.67 169.16 1192 1192 0 0 1.20 302.08 1215 1215 0 0 0.40 101.36 1223 1223 0 0 0.28 69.72 1252 1252 0 0 0.78 197.12 1364 1364 0 0 0.30 76.77 1370 PALMITIC ACID TMS 335 821 0.24 60.76 1389 289, ND3031 2073 678 1.49 377.32 1417 PENTANEDIOIC ACID, 3,3-DIMETHYL-, BIS-TMS-EST 260 418 0.50 125.53 1427 1427 0 0 0.55 138.13 1443 URIC ACID TETRA-TMS 1505 780 0.25 61.93 1462 1462 0 0 1.15 291.01 1492 PARA-HYDROXYPHENYLACETIC GLYCINE CONJ TR 2299 991 7.19 1816.50 1500 1481, NU3091 2124 782 8.74 2207.43 1596 PSEUDO URIDINE PENTA-TMS 1779 768 8.67 2189.48 1628 1472, VST031 2031 737 0.25 63.50 1746 SUCROSE OCTA-TMS 1080 924 1.05 265.38 *The named compound matches the sample peak with a reliability given by “FIT”/1000

TABLE 25 QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS FRACTION VII, BEAR URINE JZ4021: 2 mM/M Nrml CREATININE Range Organic Acids LACTIC ACID 2166  0-75 PYRUVIC ACID 211  0-20 GLYCOLIC ACID 24  0-50 ALPHA-OH-BUTYRIC 3.7 0-1 OXALIC 0.0  0-25 4-OH-BUTYRIC 0.0 0-1 HEXANOIC ACID 7.4  0-11 5-HYDROXYCAPROIC 0.0 0-1 OCTANOIC 0.0 0-1 BETA-LACTATE 10.3 0-8 SUCCINIC ACID 7  0-20 GLUTARIC ACID 0.0 0-2 2-OXO-GLUTARATE 0  0-210 FUMARIC 6.4 0-5 MALEIC 0.0 0 MALIC ACID 0.0 0-2 ADIPIC ACID 55.2 0-7 SUBERIC ACID 0.0  0-11 SEBACIC ACID 0.0 0-2 GLYCERIC ACID 0 0-4 BETA-OH-BUTYRIC 15 0-3 METHYLSUCCINIC 2082.5 0 METHYLMALONIC 0 0-5 ETHYLMALONIC 1711.8 0-4 HOMOGENTISIC ACID 14.6 0-1 PHENYLPYRUVIC ACID 3.4 0-1 SUCCINYLACETONE 10.4 0-1 3-OH-ISOVALERIC 0.6  0-21 PHOSPHATE 208   0-3000 CITRIC ACID 58  0-450 HIPPURIC ACID 48   0-2000 URIC ACID 3  0-360 Nutritionals KYNURENIC ACID 0.0 FORMIMINOGLUTAMIC 0.00 0-3 4-PYRIDOXIC ACID 0.0 0-9 PANTOTHENIC ACID 0  0-30 XANTHURENIC ACID 0.0 0-1 KYNURENINE 4.8 0-1 QUINOLINIC 0.0 0-6 OROTIC ACID 0.00 0-3 D-AM LEVULINIC 274.3  0-18 3-METHYL HISTIDINE 0  0-75 NIACINAMIDE 0.0 0-1 PSEUDOURIDINE 8927  10-220 2-DEOXYTETRONIC 0  0-75 P-HO-PHEN-ACETIC 9  0-12 XANTHINE 0  0-18 UROCANIC ACID 11 0-3 ASCORBIC ACID 0  0-160 GLYCEROL 470 0-9 Neurotransmitters GABA 0.0 0-1 HOMOVANILLIC ACID 91.0  0-10 NORMETANEPHRINE 0.7 0-1 VANILLYLMANDELIC 0.4 0-6 METANEPHRINE 0.4 0-2 5-HIAA 3.2 0-6 MHPG 0.0 0-1 ETHANOLAMINE 218 10-90 Carbohydrates THREITOL 0  0-40 ERYTHRITOL 4  0-55 ARABINOSE 0  0-30 FRUCTOSE 0.0  0-12 FUCOSE 0.0  0-12 RIBOSE 0  0-12 XYLOSE 71  0-115 GLUCOSE 101  0-110 GALACTOSE 1  0-200 MANNOSE 36  0-70 N-AC-GLUCOSAMINE 0.9 0-3 LACTOSE 107  0-60 MALTOSE 61  0-40 XYLITOL 0.0  0-15 ARABINITOL 0.0  0-30 RIBITOL 0.0  0-10 ALLOSE 0.0  0-10 GLUCURONIC ACID 35.8  0-50 GALACTONIC ACID 10  0-60 GLUCONIC ACID 4.5  0-35 GLUCARIC 0.0 0-5 MANNITOL 12.7  0-15 DULCITOL 1.0  0-10 SORBITOL 12.7  0-10 INOSITOL 2.0  0-12 SUCROSE 577  0-75 Amino Acids and Glycine Conjugates PROPIONYL GLY 0.0 0-1 BUTYRYL GLYCINE 0.0 0-1 HEXANOL GLYCINE 0.0 0-1 PHENYL PROP GLY 0.0 0-1 SUBERYL GLYCINE 0.0 0-1 ISOVALERYL GLY 279.7 0-1 TIGLY GLY 53.2 0-1 BETA MET CROT GLY 0.0 0-1 GLYCINE 584  0-500 ALANINE 437  0-130 SARCOSINE 5.2 0-8 BETA-ALANINE 0.0 0-2 B-AMINOISOBUTYRIC 2  0-50 SERINE 675  0-85 PROLINE 55.3 0-8 HYDROXY PROLINE 386  0-75 HYDROXY LYSINE 0.0 0-1 ASPARTIC ACID 96.5 0-2 ASPARAGINE 0.0 0-2 N-AC ASPARTIC 10.3  0-20 ORNITHINE 55.4 0-5 GLUTAMIC ACID 20.1 0-6 GLUTAMINE 0  0-210 PIPECOLIC ACID 0.0 0-1 LEUCINE 54.5 0-9 KETO LEUCINE 64.7 0-1 VALINE 112.8  0-18 KETO-VALINE 0.0 0-1 ISOLEUCINE 41.7 0-5 KETO-ISOLEUCINE 0.0 0-1 LYSINE 14  0-35 HISTIDINE 5  0-225 THREONINE 96  0-45 HOMOSERINE 0.0 0-1 METHIONINE 32.3 0-3 CYSTEINE 713  0-160 HOMECYSTEINE 0.0 0-1 CYSTATHIONINE 0.0 0-1 HOMOCYSTINE 0.0 0-1 CYSTINE 0.0 0-5 PHENYLALANINE 19  0-20 TYROSINE 23  0-22 TRYPTOPHAN 8  0-25 This sample contained 0.02 uMoles Creatinine/1.00 ml.

TABLE 26 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS FRACTION VII, BEAR URINE JZ4021 CONCENTRATION: THIS SAMPLE CONTAINED 0.02 mM CREATININE/mL LIB FIT AREA PEAK CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY vs1000 AREA % OF CREAT 8 10, STNO31 1893 854 4.82 564.34 20 16, 011031 1989 819 6.98 817.58 35 35, JZ4011 2300 945 0.97 113.26 58 49, AK2011 2047 821 0.68 79.19 67 SILANE, TRIMETHYLPHENOXY- 1122 935 2.89 338.68 73 1,3 PROPANEDIOL DI-TMS 1675 931 6.05 708.72 78 LACTIC ACID DI-TMS 1510 931 1.23 144.38 108 107, JZ4011 2301 889 0.78 91.61 118 104, NJ3031 2131 880 11.50 1346.76 122 119, JQ4011 2243 920 1.13 131.83 186 BETA-LACTATE DI-TMS 1654 769 2.12 248.66 190 2-METHYL 2-HYDROXY BUTYRIC ACID DI-TMS 140 887 0.43 50.10 292 UREA DI-TMS 37 813 2.61 305.69 362 TRIMETHYLSILYL ETHER OF GLYCEROL 273 913 1.73 202.95 427 METHYLSUCCINIC ACID DI-TMS 173 943 1.52 178.04 501 501 0 0 1.45 170.19 697 697 0 0 1.05 123.17 750 697, JZ4021 2316 603 0.65 76.67 809 METHYL D3 CREATININE TRI-TMS 1466 683 26.41 3094.26 848 848 0 0 0.52 60.54 985 985 0 0 0.72 84.59 1239 P-HYDROXYPHENYL LACTIC ACID TRI-TMS 578 957 5.50 644.36 1496 1481, NU3091 2124 753 0.48 56.26 1596 PSEUDO URIDINE PENTA-TMS 1779 783 9.00 1054.48 1642 1631, MI 5041 1802 789 9.19 1076.96 1689 1689 0 0 0.58 67.59 1741 TREHALOSE PER-TMS 1850 773 2.86 335.16 1746 SUCROSE OCTA-TMS 1080 923 0.97 113.28 *The named compound matches the sample peak with a reliability given by “FIT”/1000.

Isolated compounds obtained from (GC/MS were then compared to a database of chemical mass spectra for identification. Tables 21, 23, and 25 list the identified organic acids, nutritionals, carbohydrates, neurotransmitters, amino acids, and glycine conjugates of Fractions V, VI, and VII respectively.

Tables 22, 24, and 26 list peaks found in Fractions V, VI, and VII. The peaks are identified by retention time and correlated with the best match identified from the database library. Values of 700 or higher (1000 represents a perfect match) are considered indicative of substance identification. Peaks identified solely by a special number (peak #7 in Table 22 of Fraction V) indicate that this particular substance has been previously identified but that its chemical structure is unknown. When the peak number and the best match from the library are the same (as for peaks 878, 940, 1284, and 1594 in Table 22), it is an indication that these substances have not been identified by previous users of the database library. Similar data for Fractions I, II, IV, VIII, IX and X are in the following Tables 27 through 38.

BHB is found mainly in Fraction IV; MNC is found in Fractions V and VI. The most potent stimulators of osteoblast activity are found in Fractions V, VI, and VII.

SUMMARY

-   1. Separation techniques of BDI have been refined. BDI has been     separated into ten small fractions. Fractions V, VI, and VII of BDI     contain substances that produce the most potent stimulation of     osteoblasts. The substances that most strongly inhibit osteoblast     function are found in Fraction III of BDI. -   2. MNC is found in two fractions of BDI that produce the most potent     stimulation of osteoblasts—Fractions V and Fraction VI. Preliminary     data suggest that one or more components of MNC are found in     Fraction VII. -   3. The presence of known and unknown substances contained in all ten     fractions has been recorded by GC/MS.

TABLE 27 QUANTIFIED TARGET PANEL METABOLIC SCREENING LABORATORY FRACTION I, BEAR URINE JZ4041: 3 Nrml uM/L* Range Organic Acids LACTIC ACID 283233 PYRUVIC ACID 8387 GLYCOLIC ACID 1032 ALPHA-OH-BUTYRIC 19.5 OXALIC 0.0 4-OH-BUTYRIC 0.0 HEXANOIC ACID 227.5 5-HYDROXYCAPROIC 0.0 OCTANOIC 0.0 BETA-LACTATE 674.0 SUCCINIC ACID 0 GLUTARIC ACID 0.0 2-OXO-GLUTARATE 0.0 FUMARIC 35.0 MALEIC 0.0 MALIC ACID 0.0 ADIPIC ACID 49.5 SUBERIC ACID 47.5 SEBACIC ACID 0.0 GLYCERIC ACID 0.0 BETA-OH-BUTYRIC 2075.5 METHYLSUCCINIC 0.0 METHYLMALONIC 0.0 ETHYLMALONI 0.0 HOMOGENTISIC ACID 0.0 PHENYLPYRUVIC ACID 0.0 SUCCINYLACETONE 0.0 3-OH-ISOVALERIC 0.0 PHOSPHATE 3.71 mg/dL CITRIC ACID 61 HIPPURIC ACID 0 URIC ACID 1.20 mg/dL Nutritionals FORMIMINOGLUTAMIC 0.00 4-PYRIDOXIC ACID 0.0 PANTOTHENIC ACID 0.0 XANTHURENIC ACID 0.0 KYNURENINE 0.0 QUINOLINIC 0.0 OROTIC ACID 0.0 D-AM LEVULINIC ******** 3-METHYL HISTIDINE 0.00 NIACINAMIDE 0.0 PSEUDOURIDINE 221791 2-DEOXYTETRONIC 0 P-HO-PHEN-ACETIC 10 XANTHINE 0 UROCANIC ACID 96 ASCORBIC ACID 0 GLYCEROL 5903.5 Carbohydrates THREITOL 0 ERYTHRITOL 27 ARABINOSE 0 FUCOSE 0.0 RIBOSE 0.0 XYLOSE 13 FRUCTOSE 1067 GLUCOSE 35 GALACTOSE 104 MANNOSE 988 N-AC-GLUCOSAMINE 0.0 LACTOSE 2921 MALTOSE 2684 XYLITOL 0.0 ARABINITOL 0.0 RIBITOL 0.0 ALLOSE 0.0 GLUCURONIC ACID 0.0 GALACTONIC ACID 440 GLUCONIC ACID 0.0 GLUCARIC 0.0 MANNITOL 681.5 DULCITOL 91.0 SORBITOL 681.0 INOSITOL 107.0 SUCROSE 12380 Neurotransmitters GABA 89.5 HOMOVANILLIC ACID 0.0 NORMETANEPHRINE 0.0 VANILLYLMANDELIC 0.0 METANEPHRINE 0.0 5-HIAA 0.0 MHPG 0.0 ETHANOLAMINE 4416 Amino Acids and Glycine Conjugates PROPIONYL GLY 0.0 BUTYRYL GLYCINE 0.0 HEXANOL GLYCINE 0.0 PHENYL PROP GLY 0.0 SUBERYL GLYCINE 0.0 ISOVALERYL GLY 0.0 TIGLY GLY 0.0 BETA MET CROT GLY 0.0 GLYCINE 10411 ALANINE 93 SARCOSINE 108.0 BETA-ALANINE 0.0 B-AMINOISOBUTYRIC 0 SERINE 10329 PROLINE 1125.5 HYDROXY PROLINE 10671 HYDROXY LYSINE 0.0 ASPARTIC ACID 1012.0 ASPARAGINE 27.0 N-AC ASPARTIC 116.0 ORNITHINE 390.0 GLUTAMIC ACID 343.5 GLUTAMINE 0 PIPECOLIC ACID 0.0 LEUCINE 1342.0 KETO LEUCINE 2776.0 VALINE 2256.0 KETO-VALINE 0.0 ISOLEUCINE 985.0 KETO-ISOLEUCINE 0.0 LYSINE 63 HISTIDINE 0 THREONINE 771 HOMOSERINE 0.0 METHIONINE 0.0 CYSTEINE 3314.5 HOMECYSTEINE 0.0 CYSTATHIONINE 0.0 HOMOCYSTINE 0.0 CYSTINE 0.0 PHENYLALANINE 308.5 TYROSINE 370 TRYPTOPHAN 28.0 This sample contained 7.61 mg Creatinine/dL.

TABLE 28 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS FRACTION I, BEAR URINE JZ4041 CONCENTRATION: THIS SAMPLE CONTAINED 0.00 uM CREATININE/mL LIB FIT AREA % PEAK CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY vs 1000 AREA % OF CREAT 9 10, STN031 1893 849 12.44 50748.26 20 10, M13011 1782 755 12.97 52398.66 35 35, JZ4011 2300 942 1.24 5069.15 58 49, AK2011 2047 804 1.01 4129.25 67 SILANE, TRIMETHYLPHENOXY- 1122 934 3.83 15642.15 72 ETHYL AMINE DI-TMS 22 546 12.80 52202.81 79 LACTIC ACID DI-TMS 1510 959 7.49 30555.24 108 107, JZ4011 2301 939 0.99 4047.10 118 104, NJ3031 2131 882 16.86 68779.39 122 119, JQ4011 2243 930 1.60 6511.24 186 BETA-LACTATE DI-TMS 1654 770 2.91 11857.41 288 UREA DI-TMS 37 816 0.90 3654.45 361 TRIMETHYLSILYL ETHER OF GLYCEROL 273 911 1.17 4787.66 539 539 0 0 0.65 2647.54 807 METHYL D3 CREATININE TRI-TMS 1466 706 18.22 74308.42 1370 PALMITIC ACID TMS 335 857 0.92 3734.21 1519 STEARIC ACID TMS 434 870 0.70 2849.90 1595 PSEUDO URIDINE PENTA-TMS 1779 750 13.13 53567.98 1672 1669, P17031 1984 908 1.15 4703.70 1745 SUCROSE OCTA-TMS 1080 912 1.46 5942.59 *The named compound matches the sample peak with a reliability given by “FIT”/1000.

TABLE 29 QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS FRACTION II, BEAR URINE JZ4051: 4 mM/M Nrml CREATININE Range Organic Acids LACTIC ACID 94  0-75 PYRUVIC ACID 6  0-20 GLYCOLIC ACID 2  0-50 ALPHA-OH-BUTYRIC 0.1 0-1 OXALIC 0.0  0-25 4-OH-BUTYRIC 0.0 0-1 HEXANOIC ACID 0.0  0-11 5-HYDROXYCAPROIC 0.0 0-1 OCTANOIC 0.0 0-1 BETA-LACTATE 0.0 0-8 SUCCINIC ACID 3  0-20 GLUTARIC ACID 0.0 0-2 2-OXO-GLUTARATE 0  0-210 FUMARIC 0.0 0-5 MALEIC 0.0 0 MALIC ACID 0.0 0-2 ADIPIC ACID 0.0 0-7 SUBERIC ACID 0.0  0-11 SEBACIC ACID 0.0 0-2 GLYCERIC ACID 0 0-4 BETA-OH-BUTYRIC 1 0-3 METHYLSUCCINIC 0.0 0 METHYLMALONIC 0 0-5 ETHYLMALONI 0.0 0-4 HOMOGENTISIC ACID 0.0 0-1 PHENYLPYRUVIC ACID 0.7 0-1 SUCCINYLACETONE 0.0 0-1 3-OH-ISOVALERIC 0.0  0-21 PHOSPHATE 137   0-3000 CITRIC ACID 0  0-450 HIPPURIC ACID 13   0-2000 URIC ACID 0  0-360 Nutritionals KYNURENIC ACID 0.0 FORMIMINOGLUTAMIC 0.00 0-3 4-PYRIDOXIC ACID 0.0 0-9 PANTOTHENIC ACID 0  0-30 XANTHURENIC ACID 0.0 0-1 KYNURENINE 0.0 0-1 QUINOLINIC 0.0 0-6 OROTIC ACID 0.00 0-3 D-AM LEVULINIC 1.0  0-18 3-METHYL HISTIDINE 7  0-75 NIACINAMIDE 0.0 0-1 PSEUDOURIDINE 170  10-220 2-DEOXYTETRONIC 0  0-75 P-HO-PHEN-ACETIC 5  0-12 XANTHINE 0  0-18 UROCANIC ACID 0 0-3 ASCORBIC ACID 0  0-160 GLYCEROL 3 0-9 Carbohydrates THREITOL 1  0-40 ERYTHRITOL 5  0-55 ARABINOSE 0  0-30 FUCOSE 0.0  0-12 RIBOSE 0.0  0-12 XYLOSE 0  0-70 FRUCTOSE 0  0-115 GLUCOSE 2  0-110 GALACTOSE 0  0-200 MANNOSE 0  0-70 N-AC-GLUCOSAMINE 0.0 0-3 LACTOSE 1  0-60 MALTOSE 1  0-40 XYLITOL 0.9  0-15 ARABINITOL 0.0  0-30 RIBITOL 0.0  0-10 ALLOSE 0.4  0-10 GLUCURONIC ACID 0.0  0-50 GALACTONIC ACID 0  0-60 GLUCONIC ACID 0.0  0-35 CLUCARIC 0.0 0-5 MANNITOL 0.1  0-15 DULCITOL 0.1  0-10 SORBITOL 0.9  0-10 INOSITOL 0.1  0-12 SUCROSE 4  0-75 Neurotransmitters GABA 0.0 0-1 HOMOVANILLIC ACID 1.1  0-10 NORMETANEPHRINE 0.0 0-1 VANILLYLMANDELIC 0.0 0-6 METANEPHRINE 0.2 0-2 5-HIAA 1.9 0-6 MHPG 0.0 0-1 ETHANOLAMINE 6 10-90 Amino Acids and Glycine Conjugates PROPIONYL GLY 0.0 0-1 BUTYRYL GLYCINE 0.0 0-1 HEXANOL GLYCINE 0.0 0-1 PHENYL PROP GLY 0.0 0-1 SUBERYL GLYCINE 0.0 0-1 ISOVALERYL GLY 0.0 0-1 TIGLY GLY 0.0 0-1 BETA MET CROT GLY 0.0 0-1 GLYCINE 10  0-500 ALANINE 0  0-130 SARCOSINE 0.2 0-8 BETA-ALANINE 0.0 0-2 B-AMINOISOBUTYRIC 0  0-50 SERINE 9  0-85 PROLINE 0.7 0-8 HYDROXY PROLINE 13  0-75 HYDROXY LYSINE 0.0 0-1 ASPARTIC ACID 0.6 0-2 ASPARAGINE 0.0 0-2 N-AC ASPARTIC 0.0  0-20 ORNITHINE 0.1 0-5 GLUTAMIC ACID 0.5 0-6 GLUTAMINE 0  0-210 PIPECOLIC ACID 0.0 0-1 LEUCINE 0.9 0-9 KETO LEUCINE 13.4 0-1 VALINE 1.6  0-18 KETO-VALINE 0.0 0-1 ISOLEUCINE 0.5 0-5 KETO-ISOLEUCINE 0.0 0-1 LYSINE 4  0-35 HISTIDINE 0  0-225 THREONINE 0  0-45 HOMOSERINE 0.0 0-1 METHIONINE 0.0 0-3 CYSTEINE 9  0-160 HOMOCYSTEINE 0.0 0-1 CYSTATHIONINE 0.0 0-1 HOMOCYSTINE 0.0 0-1 CYSTINE 0.0 0-5 PHENYLALANINE 0  0-20 TYROSINE 0  0-22 TRYPTOPHAN 0  0-25 This sample contained 0.42 uMoles Creatinine/1.00 ml.

TABLE 30 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS FRACTION II, BEAR URINE JZ4051 CONCENTRATION: THIS SAMPLE CONTAINED 0.42 uM CREATININE/mL LIB FIT AREA % PEAK CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY vs 1000 AREA % OF CREAT 6 10, STN031 1893 823 2.11 13.22 13 13 0 0 0.53 3.32 18 16, O11031 1989 785 6.94 43.44 33 35, JZ4011 2300 882 0.59 3.70 56 49, AK2011 2047 831 0.51 3.19 65 SILANE, TRIMETHYLPHENOXY 1122 935 1.87 11.73 69 ETHYL AMINE DI-TMS 22 581 3.36 34.84 76 LACTIC ACID DI-TMS 1510 946 1.02 6.42 106 107, JZ4011 2301 785 0.58 3.62 116 104, NJ3031 2131 366 9.15 57.29 120 119, JQ4011 2243 913 0.75 4.71 184 BETA-LACTATE DI-TMS 1654 764 1.45 9.07 250 251, JZ4011 2302 923 0.47 2.97 282 UREA DI-TMS 37 721 0.83 5.23 308 283 NF3091 2093 745 18.17 113.79 354 PHOSPHATE TRI-TMS 1413 905 3.37 21.13 537 539 JZ4041 2320 956 0.56 3.53 810 CREATININE TRI-TMS 1784 946 35.05 219.48 846 3-PHENYL LACTIC TMS 2 1562 677 0.43 2.70 916 PARA-HYDROXYPHENYLACETIC ACID DI-TMS 1485 938 0.64 3.99 1189 1189 0 0 0.59 3.70 1204 1189, NU3061 2118 711 1.81 11.34 1230 MOUSE HORMONE? 1508 712 0.39 2.44 1234 1234, JD2031 2002 789 0.85 5.32 1261 STEROID M 1509 788 0.73 4.60 1369 PALMITIC ACID TMS 335 862 1.00 6.25 1519 STEARIC ACID TMS 434 918 0.38 2.38 1594 PSEUDO URIDINE PENTA-TMS 1779 816 5.75 36.03 *The named compound matches the sample peak with a reliability given by “FIT”/1000.

TABLE 31 QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS FRACTION IV, BEAR URINE JZ4071: 6 mM/M Nrml CREATININE Range Organic Acids LACTIC ACID 2393  0-75 PYRUVIC ACID 15  0-20 GLYCOLIC ACID 4  0-50 ALPHA-OH-BUTYRIC 0.7 0-1 OXALIC 0.0  0-25 4-OH-BUTYRIC 0.0 0-1 HEXANOIC ACID 28.1  0-11 5-HYDROXYCAPROIC 0.0 0-1 OCTANOIC 0.0 0-1 BETA-LACTATE 19.9 0-8 SUCCINIC ACID 1916  0-20 GLUTARIC ACID 0.0 0-2 2-OXO-GLUTARATE 210  0-210 FUMARIC 1.7 0-5 MALEIC 25.6 0 MALIC ACID 39.4 0-2 ADIPIC ACID 0.9 0-7 SUBERIC ACID 0.2  0-11 SEBACIC ACID 1.6 0-2 GLYCERIC ACID 0 0-4 BETA-OH-BUTYRIC 5822 0-3 METHYLSUCCINIC 0.0 0 METHYLMALONIC 0 0-5 ETHYLMALONIC 0.0 0-4 HOMOGENTISIC ACID 0.0 0-1 PHENYLPYRUVIC ACID 1163.4 0-1 SUCCINYLACETONE 1.0 0-1 3-OH-ISOVALERIC 2.1  0-21 PHOSPHATE 135   0-3000 CITRIC ACID 8  0-450 HIPPURIC ACID 25   0-2000 URIC ACID 2  0-360 Nutritionals KYNURENIC ACID 13.8 FORMIMINOGLUTAMIC 16.80 0-3 4-PYRIDOXIC ACID 60.5 0-9 PANTOTHENIC ACID 20  0-30 XANTHURENIC ACID 0.0 0-1 KYNURENINE 3.2 0-1 QUINOLINIC 37.4 0-6 OROTIC ACID 0.00 0-3 D-AM LEVULINIC 30.8  0-18 3-METHYL HISTIDINE 9  0-75 NIACINAMIDE 12.7 0-1 PSEUDOURIDINE 19  10-220 2-DEOXYTETRONIC 2  0-75 P-HO-PHEN-ACETIC 2  0-12 XANTHINE 0  0-18 UROCANIC ACID 1 0-3 ASCORBIC ACID 3  0-160 GLYCEROL 36 0-9 Carbohydrates THREITOL 0  0-40 ERYTHRITOL 2  0-55 ARABINOSE 0  0-30 FUCOSE 1.4  0-12 RIBOSE 1.0  0-12 XYLOSE 2  0-70 FRUCTOSE 0  0-115 GLUCOSE 55  0-110 GALACTOSE 7  0-200 MANNOSE 1  0-70 N-AC-GLUCOSAMINE 0.3 0-3 LACTOSE 11  0-60 MALTOSE 11  0-40 XYLITOL 0.0  0-15 ARABINITOL 0.0  0-30 RIBITOL 0.0  0-10 ALLOSE 0.8  0-10 GLUCURONIC ACID 11.8  0-50 GALACTONIC ACID 166  0-60 GLUCONIC ACID 0.0  0-35 CLUCARIC 0.0 0-5 MANNITOL 1.2  0-15 DULCITOL 0.0  0-10 SORBITOL 1.2  0-10 INOSITOL 0.0  0-12 SUCROSE 14  0-75 Neurotransmitters GABA 4.2 0-1 HOMOVANILLIC ACID 2.0  0-10 NORMETANEPHRINE 20.2 0-1 VANILLYLMANDELIC 2.0 0-6 METANEPHRINE 0.5 0-2 5-HIAA 5.0 0-6 MHPG 2.7 0-1 ETHANOLAMINE 17 10-90 Amino Acids and Glycine Conjugates PROPIONYL GLY 322.6 0-1 BUTYRYL GLYCINE 0.4 0-1 HEXANOYL GLYCINE 0.0 0-1 PHENYL PROP GLY 0.0 0-1 SUBERYL GLYCINE 0.0 0-1 ISOVALERYL GLY 35.7 0-1 TIGLY GLY 18.7 0-1 BETA MET CROT GLY 150.5 0-1 GLYCINE 82  0-500 ALANINE 50  0-130 SARCOSINE 0.3 0-8 BETA-ALANINE 0.0 0-2 B-AMINOISOBUTYRIC 39  0-50 SERINE 54  0-85 PROLINE 4.8 0-8 HYDROXY PROLINE 92  0-75 HYDROXY LYSINE 0.0 0-1 ASPARTIC ACID 14.0 0-2 ASPARAGINE 0.3 0-2 N-AC ASPARTIC 5.0  0-20 ORNITHINE 12.0 0-5 GLUTAMIC ACID 2.4 0-6 GLUTAMINE 46  0-210 PIPECOLIC ACID 0.0 0-1 LEUCINE 47.4 0-9 KETO LEUCINE 45.3 0-1 VALINE 9.1  0-18 KETO-VALINE 0.0 0-1 ISOLEUCINE 6.3 0-5 KETO-ISOLEUCINE 0.0 0-1 LYSINE 45  0-35 HISTIDINE 9  0-225 THREONINE 6  0-45 HOMOSERINE 2.2 0-1 METHIONINE 0.0 0-3 CYSTEINE 179  0-160 HOMECYSTEINE 0.0 0-1 CYSTATHIONINE 1.2 0-1 HOMOCYSTINE 0.0 0-1 CYSTINE 0.3 0-5 PHENYLALANINE 3  0-20 TYROSINE 5  0-22 TRYPTOPHAN 238  0-25 This sample contained 0.42 uMoles Creatine/1.00 ml.

TABLE 32 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS FRACTION IV, BEAR URINE JZ4071 CONCENTRATION: THIS SAMPLE CONTAINED 0.23 uM CREATININE/mL LIB FIT AREA % PEAK CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY vs 1000 AREA % OF CREAT 20 10, M13011 1782 716 1.28 48.98 28 10, M13011 1782 821 1.18 45.14 34 35, JZ4011 2300 836 0.25 9.56 57 49, AK2011 2047 814 0.20 7.79 66 SILANE, TRIMETHYLPHENOXY- 1122 879 0.80 30.66 71 ETHYL AMINE DI-TMS 22 529 2.92 111.91 78 LACTIC ACID DI-TMS 1510 927 4.23 162.24 107 107, JZ4011 2301 865 0.25 9.47 117 104, NJ3031 2131 872 4.13 158.52 122 119, JQ4011 2243 902 0.34 13.19 187 BETA HYDROXYBUTYRIC ACID DI-TMS 1622 930 14.85 569.62 251 251, JZ4011 2302 928 0.29 10.98 283 4-HYDROXY BUTYRIC ACID DI-TMS 97 724 0.16 6.05 293 283, NF3091 2093 745 0.25 9.61 305 283, NF3091 2093 744 1.83 70.32 355 PHOSPHATE TRI-TMS 1413 898 0.43 16.33 361 TRIMETHYLSILYL ETHER OF GLYCEROL 273 882 0.63 24.21 407 SUCCINIC ACID DI-TMS 1635 892 5.26 201.56 599 PROPIONATE GLYCINE CONJUGATE DI-TMS 165 961 1.11 42.71 611 564, JJ4021 2200 742 0.28 10.77 689 CITRAMALIC ACID TRI-TMS, 675 2103 944 0.40 15.18 722 NORLEUCINE DI-TMS 1540 656 2.48 95.07 749 749 0 0 1.11 42.72 797 259, 192 TMS 1470 367 0.27 10.23 808 CREATININE TRI-TMS 1784 913 8.32 319.11 845 845 0 0 0.19 7.28 862 862 0 0 0.18 6.77 940 GLYCOLIC ACID DI-TMS 55 405 0.35 13.32 978 251, JZ4011 2302 390 0.16 6.22 985 985 0 0 2.58 98.95 997 996, GI1021 1958 790 0.24 9.35 1000 1000 0 0 0.25 9.60 1011 BETA, PHENYLPYRUVIC ACID DI-TMS 280 887 3.95 151.29 1027 1027 0 0 0.93 35.63 1037 1037 0 0 0.41 15.72 1047 1047 0 0 0.19 7.19 1064 2-HYDROXY BENZAMIDE DI-TMS 198 421 0.51 19.63 1071 1071 0 0 0.22 8.29 1079 CIS-ACONITIC ACID TRI-TMS 540 792 6.66 255.42 1093 L-GLUTAMIC ACID, N-ACETYL-N-TMS, BIS-TMS EST 587 665 0.25 9.43 1098 862, JZ4071 2344 665 0.43 16.53 1103 1103 0 0 0.52 19.81 1114 1114 0 0 0.31 12.01 1120 1071, JZ4071 2350 685 0.64 24.48 1135 1135, JZ4011 2306 868 0.57 22.01 1178 1178 0 0 0.16 6.31 1183 6-AMINO HEXANOIC ACID DI-TMS 166 537 0.41 15.79 1196 QUINOLINIC TMS 2 1564 481 1.31 50.20 1202 1202 0 0 0.55 21.09 1228 1228 0 0 4.38 167.97 1237 1,6 DIHYDRO 1-METHYL 6-OXO 3-PYRIDINECARBOXAM 63 558 4.31 165.39 1253 MANNOSE PENTA-TMS 879 901 0.28 10.68 1277 4-PYRIDOXIC ACID TRI-TMS 580 697 0.37 14.00 1294 NORVALINE DI-TMS 128 402 0.75 28.82 1300 1300 0 0 0.39 14.89 1310 NORVALINE DI-TMS 128 432 0.25 9.50 1346 P-HO PHENYL GLYCOLIC TRI-TMS 532 735 0.17 6.61 1354 MANNOSE PENTA-TMS 879 913 0.38 14.67 1382 1382 0 0 0.64 24.60 1386 GLYCINE DI-TMS 51 477 0.18 6.93 1397 1217, NC1031 1992 543 0.16 6.32 1435 1435 0 0 0.20 7.49 1443 URIC ACID TETRA-TMS 1505 674 0.33 12.63 1510 TRYPTOPHAN TRI-TMS 1965 825 2.01 77.00 1515 1515 0 0 0.99 37.86 1545 1545 0 0 0.17 6.59 1589 1-PHENYL 2-AMINO PROPANE DI-TMS 190 712 0.16 5.96 1595 PSEUSO URIDINE PENTA-TMS 1779 945 2.48 95.21 1604 1631, MI5041 1802 692 1.73 66.36 1616 1616 0 0 0.47 17.85 1631 2-PROPENOIC ACID, 2-TMS-OXY-3-1-TMS-1H-IND 618 766 1.21 46.30 1641 1624, NU3061 2120 696 2.78 106.59 1659 1659 0 0 0.60 23.09 1665 1665 0 0 0.26 10.03 1731 TREHALOSE PER-TMS 1850 685 0.25 9.50 1745 TREHALOSE PER-TMS 1850 788 0.17 5.63 *The named compound matches the sample peak with a reliability given by “FIT”/1000.

TABLE 33 QUANTIFIED TARGET PANEL URINE ORAGANIC COMPOUNDS FRACTION VIII, BEAR URINE JZ4091: 8 mM/M Nrml CREATININE Range Organic Acids LACTIC ACID 38661  0–75 PYRUVIC ACID 0  0–20 GLYCOLIC ACID 0  0–50 ALPHA-OH-BUTYRIC 0.0 0–1 OXALIC 0.0  0–25 4-OH-BUTYRIC 0.0 0–1 HEXANOIC ACID 0.0  0–11 5-HYDROXYCAPROIC 0.0 0–1 OCTANOIC 0.0 0–1 BETA-LACTATE 0.0 0–8 SUCCINIC ACID 0  0–20 GLUTARIC ACID 0.0 0–2 2-OXO-GLUTARATE 0  0–210 FUMARIC 0.0 0–5 MALEIC 0.0 0 MALIC ACID 0.0 0–2 ADIPIC ACID 3878.3 0–7 SUBERIC ACID 0.0  0–11 SEBACIC ACID 244.7 0–2 GLYCERIC ACID 0 0–4 BETA-OH-BUTYRIC 89 0–3 METHYLSUCCINIC 0.0 0 METHYLMALONIC 0 0–5 ETHYLMALONIC ******** 0–4 HOMOGENTISIC ACID 0.0 0–1 PHENYLPYRUVIC ACID 0.0 0–1 SUCCINYLACETONE 0.0 0–1 3-OH-ISOVALERIC 0.0  0–21 PHOSPHATE 317   0–3000 CITRIC ACID 37  0–450 HIPPURIC ACID 84990   0–2000 URIC ACID 125  0–360 Nutritionals KYNURENIC ACID 7544.8 FORMIMINOGLUTAMIC 0.00 0–3 4-PYRIDOXIC ACID 0.0 0–9 PANTOTHENIC ACID 0  0–30 XANTHURENIC ACID 0.0 0–1 KYNURENINE 0.0 0–1 QUINOLINIC 0.0 0–6 OROTIC ACID 0.00 0–3 D-AM LEVULINIC 0.0  0–18 3-METHYL HISTIDINE 0  0–75 NIACINAMIDE 0.0 0–1 PSEUDOURIDINE 7176  10–220 2-DEOXYTETRONIC 0  0–75 P-HO-PHEN-ACETIC 1019  0–12 XANTHINE 0  0–18 UROCANIC ACID 907 0–3 ASCORBIC ACID 0  0–160 GLYCEROL 8524 0–9 Neurotransmitters GABA 0.0 0–1 HOMOVANILLIC ACID 4038.8  0–10 NORMETANEPHRINE 0.0 0–1 VANILLYLMANDELIC 0.0 0–6 METANEPHRINE 374.2 0–2 5-HIAA 6190.5 0–6 MHPG 0.0 0–1 ETHANOLAMINE 3152 10–90 Carbohydrates THREITOL 0  0–40 ERYTHRITOL 0  0–55 ARABINOSE 0  0–30 FUCOSE 0.0  0–12 RIBOSE 0.0  0–12 XYLOSE 0  0–70 FRUCTOSE 3266  0–115 GLUCOSE 4435  0–110 GALACTOSE 5127  0–200 MANNOSE 2585  0–70 N-AC-GLUCOSAMINE 11.8 0–3 LACTOSE 4679  0–60 MALTOSE 4470  0–40 XYLITOL 0.0  0–15 ARABINITOL 0.0  0–30 RIBITOL 0.0  0–10 ALLOSE 384.7 0–10 GLUCURONIC ACID 0.0  0–50 GALACTONIC ACID 13137  0–60 GLUCONIC ACID 0.0  0–35 GLUCARIC 42.7 0–5 MANNITOL 604.1  0–15 DULCITOL 0.0  0–10 SORBITOL 603.4  0–10 INOSITOL 0.0  0–12 SUCROSE 18255  0–75 Amino Acids and Glycine Conjugates PROPIONYL GLY 0.0 0–1 BUTYRYL GLYCINE 2523.4 0–1 HEXANOL GLYCINE 0.0 0–1 PHENYL PROP GLY 0.0 0–1 SUBERYL GLYCINE 0.0 0–1 ISOVALERYL GLY ******** 0–1 TIGLY GLY 0.0 0–1 BETA MET CROT GLY ******** 0–1 GLYCINE 9496  0–500 ALANINE 7063  0–130 SARCOSINE 80.5 0–8 BETA-ALANINE 0.0 0–2 B-AMINOISOBUTYRIC 525  0–50 SERINE 10517  0–85 PROLINE 917.5 0–8 HYDROXY PROLINE 12808  0–75 HYDROXY LYSINE 1407.6 0–1 ASPARTIC ACID 1866.1 0–2 ASPARAGINE 0.0 0–2 N-AC ASPARTIC 0.0  0–20 ORNITHINE 1826.4 0–5 GLUTAMIC ACID 364.9 0–6 GLUTAMINE 0  0–210 PIPECOLIC ACID 0.0 0–1 LEUCINE 1200.1 0–9 KETO LEUCINE 913.8 0–1 VALINE 1532.7  0–18 KETO-VALINE 0.0 0–1 ISOLEUCINE 871.7 0–5 KETO-ISOLEUCINE 0.0 0–1 LYSINE 34440  0–35 HISTIDINE 1307  0–225 THREONINE 1240  0–45 HOMOSERINE 0.0 0–1 METHIONINE ******** 0–3 CYSTEINE 10527  0–160 HOMECYSTEINE 0.0 0–1 CYSTATHIONINE 0.0 0–1 HOMOCYSTINE 0.0 0–1 CYSTINE 0.0 0–5 PHENYLALANINE 896  0–20 TYROSINE 1136  0–22 TRYPTOPHAN 575  0–25 This sample contained 0.00 uMoles Creatinine/7.20 ml.

TABLE 34 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS FRACTION VIII, BEAR URINE JZ4091 CONCENTRATION: THIS SAMPLE CONTAINED 0.00 uM CREATININE/mL LIB FIT AREA % PEAK # CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY vs 1000 AREA % OF CREAT 14 13, JZ4051 2321 783 0.61 2309.70 18 13, JZ4051 2321 759 2.92 11073.36 62 SILANE, TRIMETHYLPHENOXY- 1122 877 0.63 2396.66 69 1,3 PROPANEDIOL DI-TMS 1675 925 2.01 7601.11 74 LACTIC ACID DI-TMS 1510 907 0.65 2452.00 114 104, NI3031 2131 850 3.43 12980.22 185 BETA-LACTATE DI-TMS 1654 773 0.42 1575.81 189 2-HYDROXY PENTANOIC ACID DI-TMS 141 918 1.13 4290.31 291 291 0 0 1.55 5864.71 354 DIMETHYL MALANIC ACID DI-TMS 171 954 0.82 3110.44 362 TRIMETHYLSILYL ETHER OF GLYCEROL 273 938 0.99 3754.66 622 3-METHYL 2-PENTENEDIOIC ACID DI-TMS 224 892 0.62 2366.22 682 3-METHYL BUTANOATE GLYCINE CONJUGATE TMS 74 625 0.47 1788.05 696 3-METHYL 2-PENTENDIOIC ACID DI-TMS, Z- 222 840 0.47 1778.00 752 GLYCINE, N-3-METHYL-1-OXOBUTYL-N-TMS-, TRIMET 255 942 3.62 13706.16 803 METHYL D3 CREATININE TRI-TMS 1466 743 16.38 62054.19 848 848, JZ4021 2317 887 3.09 11698.73 1104 1104 0 0 3.57 13521.55 1123 1112, M20021 1823 765 0.67 2526.55 1158 3,4-DIHYDROXY BENZENEACETIC ACID TRI-TMS 531 834 0.54 2054.74 1196 1189, JZ4051 2322 961 3.87 14654.56 1211 1189, NU3061 2118 697 19.22 72308.71 1232 L-GLUTAMIC ACID, N-ACETYL-N-TMS-, BIS-TMS EST 587 526 2.22 8414.89 1241 P-HYDROXYPHENYL LACTIC ACID TRI-TMS 578 941 9.80 37151.69 1287 HYDROXY PROLINE DI-TMS 1610 424 0.72 2710.46 1370 PALMITIC ACID TMS 335 639 1.07 4055.54 1413 1481, NU3091 2124 403 0.46 1761.13 1506 PARA-HYDROXY HIPPURIC ACID DI-TMS 377 901 1.04 3941.33 1596 PSEUDO URIDINE PENTA-TMS 1779 953 7.00 26509.32 1642 1631, M15041 1802 795 8.81 33369.32 1740 TREHALOSE PER-TMS 1850 781 0.44 1655.34 1746 SUCROSE OCTA-TMS 1080 892 1.40 5286.62 *The named compound matches the sample peak with a reliability given by “FIT”/1000.

TABLE 35 QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS FRACTION IX. BEAR URINE JZ4101: 9 mM/M Nrml CREATININE Range Organic Acids LACTIC ACID 856  0–75 PYRUVIC ACID 52  0–20 GLYCOLIC ACID 7  0–50 ALPHA-OH-BUTYRIC 1.9 0–1 OXALIC 0.0  0–25 4-OH-BUTYRIC 0.0 0–1 HEXANOIC ACID 415.0  0–11 5-HYDROXYCAPROIC 0.0 0–1 OCTANOIC 0.0 0–1 BETA-LACTATE 0.0 0–8 SUCCINIC ACID 4  0–20 GLUTARIC ACID 0.0 0–2 2-OXO-GLUTARATE 0  0–210 FUMARIC 7.1 0–5 MALEIC 0.0 0 MALIC ACID 0.0 0–2 ADIPIC ACID 33.7 0–7 SUBERIC ACID 536.8  0–11 SEBACIC ACID 1.1 0–2 GLYCERIC ACID 0 0–4 BETA-OH-BUTYRIC 12 0–3 METHYLSUCCINIC 0.0 0 METHYLMALONIC 0 0–5 ETHYLMALONIC 137.0 0–4 HOMOGENTISIC ACID 0.0 0–1 PHENYLPYRUVIC ACID 110.6 0–1 SUCCINYLACETONE 0.0 0–1 3-OH-ISOVALERIC 1.8  0–21 PHOSPHATE 317   0–3000 CITRIC ACID 136  0–450 HIPPURIC ACID 35604   0–2000 URIC ACID 4  0–360 Nutritionals KYNURENIC ACID 297.6 FORMIMINOGLUTAMIC 0.00 0–3 4-PYRIDOXIC ACID 0.0 0–9 PANTOTHENIC ACID 37  0–30 XANTHURENIC ACID 18.4 0–1 KYNURENINE 19.8 0–1 QUINOLINIC 0.0 0–6 OROTIC ACID 0.00 0–3 D-AM LEVULINIC 20.0  0–18 3-METHYL HISTIDINE 32  0–75 NIACINAMIDE 0.0 0–1 PSEUDOURIDINE 22608  10–220 2-DEOXYTETRONIC 2  0–75 P-HO-PHEN-ACETIC 18  0–12 XANTHINE 6  0–18 UROCANIC ACID 49 0–3 ASCORBIC ACID 2  0–160 GLYCEROL 352 0–9 Carbohydrates THREITOL 0  0–40 ERYTHRITOL 0  0–55 ARABINOSE 9  0–30 FUCOSE 41.0  0–12 RIBOSE 41.0  0–12 XYLOSE 3  0–70 FRUCTOSE 14  0–115 GLUCOSE 232  0–110 GALACTOSE 1239  0–200 MANNOSE 35  0–70 N-AC-GLUCOSAMINE 6.5 0–3 LACTOSE 145  0–60 MALTOSE 140  0–40 XYLITOL 0.0  0–15 ARABINITOL 0.0  0–30 RIBITOL 0.0  0–10 ALLOSE 6.4  0–10 GLUCURONIC ACID 38.1  0–50 GALACTONIC ACID 421  0–60 GLUCONIC ACID 4.9  0–35 GLUCARIC 2.9 0–5 MANNITOL 4.1  0–15 DULCITOL 1.0  0–10 SORBITOL 7.7  0–10 INOSITOL 3.9  0–12 SUCROSE 483  0–75 Neurotransmitters GABA 8.8 0–1 HOMOVANILLIC ACID 6221.3  0–10 NORMETANEPHRINE 53.6 0–1 VANILLYLMANDELIC 30.3 0–6 METANEPHRINE 156.8 0–2 5-HIAA 4791.4 0–6 MHPG 0.0 0–1 ETHANOLAMINE 211 10–90 Amino Acids and Glycine Conjugates PROPIONYL GLY 8.7 0–1 BUTYRYL GLYCINE 0.0 0–1 HEXANOYL GLYCINE 39.1 0–1 PHENYL PROP GLY 0.0 0–1 SUBERYL GLYCINE 0.3 0–1 ISOVALERYL GLY 1852.0 0–1 TIGLY GLY 4.7 0–1 BETA MET CROT GLY 36.8 0–1 GLYCINE 614  0–500 ALANINE 3  0–130 SARCOSINE 1.2 0–8 BETA-ALANINE 0.0 0–2 B-AMINOISOBUTYRIC 232  0–50 SERINE 403  0–85 PROLINE 35.4 0–8 HYDROXY PROLINE 1036  0–75 HYDROXY LYSINE 14.3 0–1 ASPARTIC ACID 105.0 0–2 ASPARAGINE 0.6 0–2 N-AC ASPARTIC 41.4  0–20 ORNITHINE 153.8 0–5 GLUTAMIC ACID 53.2 0–6 GLUTAMINE 40  0–210 PIPECOLIC ACID 0.0 0–1 LEUCINE 62.3 0–9 KETO LEUCINE 533.3 0–1 VALINE 60.8  0–18 KETO-VALINE 0.0 0–1 ISOLEUCINE 49.9 0–5 KETO-ISOLEUCINE 0.0 0–1 LYSINE 16777  0–35 HISTIDINE 452  0–225 THREONINE 69  0–45 HOMOSERINE 0.0 0–1 METHIONINE 254.1 0–3 CYSTEINE 2504  0–160 HOMOCYSTEINE 0.0 0–1 CYSTATHIONINE 0.5 0–1 HOMOCYSTINE 4.3 0–1 CYSTINE 16.5 0–5 PHENYLALANINE 216  0–20 TYROSINE 73  0–22 TRYPTOPHAN 404  0–25 This sample contained 0.02 uMoles Creatinine/7.20 ml.

TABLE 36 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS FRACTION IX, BEAR URINE JZ4101 CONCENTRATION: THIS SAMPLE CONTAINED 0.00 μM CREATININE/mL LIB FIT AREA % PEAK # CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY vs 1000 AREA % OF CREAT 7 6, JI4081 2189 745 0.67 179.37 10 13, JZ4051 2321 739 0.18 47.76 19 13, AK2011 2044 737 1.17 312.52 66 SILANE, TRIMETHYLPHENOXY- 1122 896 0.29 77.67 71 ETHYL AMINE DI-TMS 22 549 1.79 479.46 78 PROPENE GLYCOL DI-TMS 50 922 0.16 41.63 107 107, JZ4011 2301 849 0.14 37.91 117 104, NJ3031 2131 851 3.34 897.03 122 119, JQ4011 2243 902 0.13 34.73 186 BETA-LACTATE DI-TMS 1654 777 0.41 110.10 293 2-HYDROXY HEXANOIC ACID DI-TMS 1682 784 3.73 1000.76 362 TRIMETHYLSILYL ETHER OF GLYCEROL 273 909 0.50 134.35 383 SILANE, TRIMETHYL 1-METHYLBUTOXY- 1112 493 0.11 30.42 540 539, JZ4041 2320 930 0.29 78.34 613 613 0 0 0.24 63.30 622 3-METHYL 2-PENTENEDIOIC ACID DI-TMS 224 833 0.33 88.13 642 613, JZ4041 2370 711 1.24 332.62 687 BENZENEACETIC, ACID ALPHA - TMS-OXY, -TRIM 246 889 1.24 332.20 696 3-METHYL 2-PENTENDIOIC ACID DI-TMS, Z- 222 891 1.16 41.93 753 HEXANEDIOIC ACID, 3-METHYL-, BIS-TMS-ESTER 258 663 1.64 440.24 781 HEXANEDIOIC ACID, 3-METHYL-, BIS-TMS-ESTER 258 793 0.18 49.23 798 METHYL D3 CREATININE TRI-TMS 1466 717 0.11 30.11 809 METHYL D3 CREATININE TRI-TMS 1466 701 12.34 3310.78 821 ORTHO-HYDROXYPHENYLACETIC ACID DI-TMS 247 929 0.60 161.70 852 2-HYDROXY 3-PHENYL PROPIONIC ACID DI-TMS 287 921 7.95 2132.51 861 848, JZ4021 2317 685 0.18 47.45 879 HEPTANEDIOIC ACID, BIS-TMS-ESTER 259 905 1.33 355.68 903 PARA-HYDROXY BENZOIC DI-TMS 202 868 0.45 119.54 913 PARA-HYDROXYPHENYLACETIC ACID, DI-TMS 1485 927 0.13 35.95 925 PARA-HYDROXYPHENYLACETIC ACID-DI-TMS 1485 835 13.82 3707.87 930 938, DQ3041 2164 757 0.10 28.08 975 975 0 0 1.18 316.77 986 985, JZ4021 2318 899 0.29 78.99 991 991 0 0 0.15 38.94 1001 OCTANEDIOIC ACID, BIS-TMS-ESTER 306 744 0.36 95.83 1087 HOMOVANILLIC ACID DI-TMS 331 946 2.49 667.03 1103 1104, JZ4091 2369 930 0.43 114.58 1116 1116 0 0 0.53 142.93 1125 1112, M20021 1823 763 4.82 1292.51 1146 HIPPORIC ACID TMS ESTER 103 903 1.02 273.29 1184 1189, JZ4051 2322 954 0.31 82.08 1192 1189, JZ4051 2322 890 0.33 89.21 1200 1189, NU3061 2118 705 0.72 194.06 1211 1189, NU3061 2118 704 5.65 1515.93 1234 L-GLYTAMIC ACID, N-ACETYL-N-TMS-, BIS-TMS EST 587 494 3.37 902.66 1243 P-HYDROXYPHENYL, LACTIC ACID, TRI-TMS 578 951 0.75 201.16 1259 PROPANEDIOIC ACID, TMS-OXY-, BIS-TMS-ESTER 594 238 0.52 139.80 1273 HYDROXY PROLINE DI-TMS 1610 349 0.17 46.73 1280 1H-INDOLE-2-CARBOXYLIC ACID, 5-ETHYL-1-TMS- 343 646 0.29 76.62 1289 991, JZ4101 2372 460 1.53 409.12 1332 1332 0 0 0.13 35.00 1354 1354 0 0 0.13 35.22 1364 MANNO-ONIC ACID, LACTONE TETRA-TMS 732 454 0.30 81.30 1371 PALMITIC ACID TMS 335 670 0.91 245.18 1414 1481, NU3091 2124 464 0.60 160.27 1426 SILANE, TRIMETHYL 3-PHENYLPROPOXY- 1158 500 0.19 50.80 1451 BETA AMINO BUTYRIC ACID DI-TMS 89 761 0.22 58.41 1481 TRYPIOPHAN TRI-TMS 1965 477 0.55 146.22 1486 1472, VST031 2031 771 4.74 1271.10 1509 5-HYDROXY INDOLE ACETIC ACID TRI-TMS 592 943 3.19 856.94 1520 STEARIC ACID TMS 434 787 0.14 36.94 1573 6-HYDROXY-HEPTANOIC DI-TMS 1690 275 0.30 79.25 1596 PSEUDO URIDINE PENTA-TMS 1779 746 5.92 1587.71 1628 1472, VST031 2031 799 0.26 69.56 1641 1631, MI5041 1802 826 0.87 234.00 1673 1472, VST031 2031 650 1.73 464.94 1680 1676, JD2011 2001 624 0.33 87.91 1746 SUCROSE OCTA-TMS 1080 847 0.31 83.03 *The named compound matches the sample peak with a reliability given by “FIT”/1000.

TABLE 37 QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS FRACTION X, BEAR URINE JZ4111: 8 mM/M Nrml CREATININE Range Organic Acids LACTIC ACID 19433  0–75 PYRUVIC ACID 950  0–20 GLYCOLIC ACID 196  0–50 ALPHA-OH-BUTYRIC 14.8 0–1 OXALIC 36.0  0–25 4-OH-BUTYRIC 0.0 0–1 HEXANOIC ACID 60.0  0–11 5-HYDROXYCAPROIC 12.6 0–1 OCTANOIC 37.4 0–1 BETA-LACTATE 234.1 0–8 SUCCINIC ACID 135  0–20 GLUTARIC ACID 0.0 0–2 2-OXO-GLUTARATE 0  0–210 FUMARIC 21.9 0–5 MALEIC 0.0 0 MALIC ACID 18.8 0–2 ADIPIC ACID 30.4 0–7 SUBERIC ACID 4707.2  0–11 SEBACIC ACID 3.0 0–2 GLYCERIC ACID 30 0–4 BETA-OH-BUTYRIC 321 0–3 METHYLSUCCINIC 0.0 0 METHYLMALONIC 0 0–5 ETHYLMALONI 103.0 0–4 HOMOGENTISIC ACID 0.0 0–1 PHENYLPYRUVIC ACID 347.5 0–1 SUCCINYLACETONE 2.2 0–1 3-OH-ISOVALERIC 1.8 0–21 PHOSPHATE 814   0–3000 CITRIC ACID 46  0–450 HIPPURIC ACID 5949   0–2000 URIC ACID 40  0–360 Nutritionals KYNURENIC ACID 6.2 FORMIMINOGLUTAMIC 0.60 0–3 4-PYRIDOXIC ACID 0.0 0–9 PANTOTHENIC ACID 3  0–30 XANTHRENIC ACID 2.6 0–1 KYNURENINE 70.3 0–1 QUINOLINIC 0.0 0–6 DROTIC ACID 28.54 0–3 D-AM LEVULINIC 541.3  0–18 3-METHYL HISTIDINE 216  0–75 NIACINAMIDE 62.7 0–1 PSEUDOURIDINE 10351  10–220 2-DEOXYTETRONIC 41  0–75 P-HO-PHEN-ACETIC 254  0–12 XANTHINE 14  0–18 UROCANIC ACID 255 0–3 ASCORBIC ACID 1  0–160 GLYCEROL 11477 0–9 Carbohydrates THREITOL 7  0–40 ERYTHRITOL 7  0–55 ARABINOSE 25  0–30 FUCOSE 379.6  0–12 RIBOSE 219.1  0–12 XYLOSE 8  0–70 FRUCTOSE 808  0–115 GLUCOSE 432  0–110 GALACTOSE 19  0–200 MANNOSE 406  0–70 N.AC-GLUCOSAMINE 28.8 0–3 LACTOSE 349  0–60 MALTOSE 237  0–40 XYLITOL 27.6  0–15 ARABINITOL 16.0  0–30 RIBITOL 0.0  0–10 ALLOSE 61.7  0–10 GLUCURONIC ACID 239.8  0–50 GALACTONIC ACID 400  0–60 GLUCONIC ACID 11.2  0–35 GLUCARIC 9.0 0–5 MANNITOL 31.5  0–15 DULCITOL 10.6  0–10 SORBITOL 55.4  0–10 INOSITOL 13.6  0–12 SUCROSE 1788  0–75 Neurotransmitters GABA 24.8 0–1 HOMOVANILLIC ACID 1673.5  0–10 NORMETANEPHRINE 17.0 0–1 VANILLYLMANDELIC 2.6 0–6 METANEPHRINE 3.1 0–2 5-HIAA 1026.9 0–6 MHPG 1.2 0–1 ETHANOLAMINE 679 10–90 Amino Acids and Glycine Conjugates PROPIONYL GLY 16.6 0–1 BUTYRYL GLYCINE 0.0 0–1 HEXANOL GLYCINE 444.9 0–1 PHENYL PROP GLY 243.3 0–1 SUBERYL GLYCINE 4.4 0–1 ISOVALERYL GLY 144.3 0–1 TIGLY GLY 5.7 0–1 BETA MET CROT GLY 353.8 0–1 GLYCINE 2601  0–500 ALANINE 1316  0–130 SARCOSINE 15.4 0–8 BETA-ALANINE 31.3 0–2 B-AMINOISOBUTYRIC 538  0–50 SERINE 2443  0–85 PROLINE 244.2 0–8 HYDROXY PROLINE 3372  0–75 HYDROXY LYSINE 127.6 0–1 ASPARTIC ACID 499.6 0–2 ASPARAGINE 0.2 0–2 N.AC ASPARTIC 13.5  0–20 ORNITHINE 442.4 0–5 GLUTAMIC ACID 6.0 0–6 GLUTAMINE 22.0  0–210 PIPECOLIC ACID 0.4 0–1 LEUCINE 337.8 0–9 KETO LEUCINE 1066.2 0–1 VALINE 417.4  0–18 KETO-VALINE 1.7 0–1 ISOLEUCINE 274.6 0–5 KETO-ISOLEUCINE 80.6 0–1 LYSINE 2599  0–35 HISTIDINE 203  0–225 THREONINE 377  0–45 HOMOSERINE 0.0 0–1 METHIONINE 20.8 0–3 CYSTEINE 3059  0–160 HOMECYSTEINE 1.0 0–1 CYSTATHIONINE 5.6 0–1 HOMOCYSTINE 59.7 0–1 CYSTINE 9.4 0–5 PHENYLALANINE 233  0–20 TYROSINE 190  0–22 TRYPTOPHAN 130  0–25 This same contianed 0.03 uMoles Creatinine/100 ml.

TABLE 38 METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUTENTS FRACTION X, BEAR URINE JZ4111 CONCENTRATION: THIS SAMPLE CONTAINED 0.03 uM CREATININE/mL LIB FIT AREA PEAK # CONSTITUENT'S BEST MATCH FROM LIBRARY* ENTRY vs 1000 AREA % OF CREAT 6 6, JI4081 2189 675 0.71 314.00 9 10, STN031 1893 719 0.65 288.12 12 13, JZ4051 2321 561 0.48 215.50 20 10, M13011 1782 719 2.07 921.84 36 35, JZ4011 2300 847 0.22 97.76 51 42, M20021 1816 726 0.19 83.08 59 49, AK2011 2047 833 0.19 83.56 68 SILANE, TRIMETHYLPHENOXY- 1122 847 0.73 324.60 72 ETHYL AMINE DI-TMS 22 513 2.08 923.09 80 LACTIC ACID DI-TMS 1510 874 1.34 594.49 88 BORATE TRI-TMS 186 618 0.06 26.27 108 107, JZ4011 2301 847 0.20 90.08 118 104, NJ3031 2131 744 2.49 1108.84 123 119, JQ4011 2243 907 0.30 131.95 166 SILANOL, TRIMETHYL-, CARBONATE 2:1 1429 647 0.07 32.24 186 BETA-LACTATE DI-TMS 1654 781 0.54 241.79 224 92, NA3011 2070 757 0.07 29.54 252 251, JZ4011 2302 848 0.09 39.70 294 4-METHYL 2-HYDROXY PETANOIC ACID DI-TMS 178 807 5.30 2356.51 297 2-HYDROXY HEXANOIC ACID DI-TMS 1682 786 3.49 1551.67 301 291, JZ4091 2368 775 1.56 693.60 336 ETHANOLAMINE TRI-TMS 181 907 0.13 59.44 349 PEAK 459, A02011 1855 511 0.06 26.28 365 TRIMETHYLSILYL ETHER OF GLYCEROL 273 824 1.90 844.99 386 TETRADECANOIC ACID TMS 251 510 0.12 52.53 398 GLYCINE TRI-TMS 1539 869 0.44 197.40 503 SERINE TRI-TMS 322 957 0.51 228.07 540 539, JZ4041 2320 886 0.37 166.09 613 613, JZ4101 2370 855 0.41 182.98 642 1364, JZ4011 2312 370 0.69 307.69 686 BENZENEACETIC ACID, ALPHA. - TMS-OXY-TRIM 246 874 0.19 83.47 753 HEXANEDIOCIC ACID, 3-METHYL-BIS-TMS-ESTER 258 758 1.53 678.67 773 SILANE, DIMETHYLPHENOXY TRIMETHYL, 1150 332 0.12 55.52 781 HEPANEDIOIC ACID, BIS-TMS-ESTER 259 624 0.14 60.31 798 METHYL D3 CREATININE TRI-TMS 1466 715 0.04 18.49 809 METHYL D3 CREATININE TRI-TMS 1466 707 4.53 2013.68 822 ORTHO-HYDROXYPHENYLACETIC ACID DI-TMS 247 907 1.04 460.14 856 2-HYDROXY 3-PHENYL PROPIONIC ACID DI-TMS 287 872 7.69 3420.08 880 HEPTANEDIOIC ACID, BIS-TMS-ESTER 259 866 0.95 420.88 907 PARA HYDROXY BENZOIC DI-TMS 202 873 4.41 1959.38 914 PARA-HYDROXYPHENYLACETIC ACID DI-TMS 1485 628 0.94 418.25 928 PARA-HYDROXYPHENYLACETIC ACID DI-TMS 1485 811 9.47 4211.72 938 1234, JZ4061 2333 444 0.07 32.28 946 HEXANOYL GLYCINE DI-TMS 1656 724 0.19 83.16 971 975, JZ4101 2371 813 0.23 100.98 976 975, JZ4101 2371 877 2.17 964.67 987 985, JZ4021 2318 756 0.18 81.73 992 991, JZ4101 2372 814 0.20 88.90 996 SUBERIC ACID DI-TMS 1633 520 0.05 21.95 1003 OCTANEDIOIC ACID, BIS-TMS-ESTER 306 726 2.12 940.43 1010 1062, NJ3051 2135 474 0.37 163.67 1015 561, LB1031 VALPROIC ACID METABOLITE, MSL 1973 527 0.55 246.28 1031 SILANE, TRIMETHYL PHENETHYLTHIO- 1161 389 0.23 102.67 1046 SEBACIC ACID, BIS-TMS-ESTER 393 612 0.36 160.75 1060 975, JZ4101 2371 704 0.04 19.97 1068 HYDROCINNAMIC ACID, P-TMS-, TRIMETHYLSILYLES 288 688 0.28 126.21 1081 1160, JG4021 2179 315 0.37 164.16 1088 1062, NJ3051 2135 770 1.35 599.54 1095 1332, JZ4101 2374 598 0.39 172.38 1103 1104, JZ4091 2369 784 0.06 26.57 1116 1116, JZ4101 2373 861 0.86 382.04 1124 1112, M20021 1823 804 0.34 149.94 1133 877, JK4071 2237 414 0.28 125.70 1138 975, JZ4101 2371 386 0.41 181.50 1145 HIPPURIC ACID TMS ESTER 103 779 0.13 59.11 1157 ORNITHINE N5, N5 TETRA-TMS 1536 836 0.13 57.72 1164 FRUCTOSE PENTA-TMS 881 660 0.18 79.07 1169 TETRADECANOIC ACID TMS 251 789 0.17 75.71 1175 METHYL ALPHA-GLUCOSIDE TETRA-TMS 790 410 0.30 134.71 1187 24, AK2011 2045 508 0.23 103.04 1199 1189, JZ4051 2322 828 3.17 1408.37 1213 1189, NU3061 2118 676 6.41 2850.85 1222 SEBACIC ACID, BIS-TMS-ESTER 393 521 0.07 31.48 1227 META-HYDROXYPHENYL ACETIC ACID DI-TMS 248 274 0.21 91.70 1234 ACETIC ACID, PHENOXY-, TRIMETHYLSILYL ESTER 66 481 0.60 265.32 1255 GALACTOSE PENTA-TMS 878 571 0.69 304.74 1263 996, JZ4061 2329 391 0.08 37.07 1279 1H-INDOLE-2-CARBOXYLIC ACID, 5-ETHYL-I-TMS-. 343 445 0.11 49.11 1288 INDOLE 2-ACETIC ACID I-TMS, TMS-ESTER 316 858 2.51 1117.19 1302 GL1021, 678 1964 451 0.32 140.3 1309 1H-INDOLE-3-ETHANAMINE, N, I-BIS-TMS-5-TMS-OX 547 565 0.27 119.16 1334 3-HYDROXYTETRADECENEDIOIC ACID I 1708 420 0.13 59.54 1344 1H-INDOLE-5-CARBOXYLIC ACID, I-TMS-, TRIMETHY 266 441 0.38 170.74 1355 D-MANNOPYRANOSE PENTA-TMS 892 905 0.43 192.76 1371 PALMITIC ACID TMS 335 892 0.77 340.90 1398 GALACTURONIC ACID PENTATMS 915 629 0.07 31.57 1406 1246, JZ4061 2334 434 0.24 108.11 1411 1032, M15041 1796 335 0.04 19.48 1423 988, NE3031 2083 407 0.13 57.19 1443 1300, JZ4071 2356 465 0.09 37.89 1455 DODECENEDIOIC ACID DI-TMS, CIS? 1695 433 0.07 31.96 1489 1472, VST031 2031 694 4.88 2167.24 1502 OLEIC ACID, TRIMETHYLSILYL ESTER. 1614 677 0.13 56.05 1509 5-HYDROXY INDOLE ACETIC ACID TRI-TMS 592 889 0.36 159.16 1520 STEARIC ACID TMS 434 728 0.55 244.65 1529 982, N03031 2142 405 0.12 53.30 1537 3-HYDROXYDODECANEDIOIC ACID-TMS-3 1776 708 0.05 20.19 1546 996, GI1021 1958 448 0.27 118.50 1558 HEPTANEDIOIC ACID, 4-OXO-, BIS-TMS ESTER 305 381 0.12 54.63 1562 1472, VST031 2031 635 0.07 32.52 1596 PSEUDO URIDINE PENTA-TMS 1779 690 2.10 933.44 1603 988, OK1041 1990 574 0.09 40.28 1609 1472, VST031 2031 552 0.04 19.08 1612 251, JZ4011 2302 365 0.06 24.80 1620 D-GALACTOSE, 2-AMINO-2-DEOXY-3,4,5,6-TETRAKIS 746 406 0.07 33.22 1628 1472, VST031 2031 729 0.55 246.19 1652 1472, VST031 2031 713 0.14 62.64 1664 1631, M15041 1802 567 0.09 41.81 1674 1669, PI7031 1984 687 2.27 1011.28 1680 1472, VST031 2031 463 0.08 33.58 1686 1189, JZ4051 2322 252 0.06 25.53 1692 1073, RT1051 2040 395 0.05 22.18 1701 2-HYDROXYTETRADECENEDIOIC ACID 1704 385 0.08 36.13 1728 533, LB1031 VALPROIC ACID METABOLITE, MSL 1972 409 0.04 19.96 1746 SUCROSE OCTA-TMS 1080 888 0.73 324.31 1795 LACTOSE OCTA-TMS 1854 785 0.08 36.36 1839 1785, YD1011 1875 414 0.06 25.81 *The named compound matches the sample peak with a reliability given by “FIT”/1000 Further Purification of MNC in Fraction VI Using HPLC

Fraction VI was further purified using HPLC. After lyophilization and reconstitution in methanol, aliquots of Fraction VI were loaded onto a HPLC using a C₁₈ column. A gradient of 0.1 M ammonium formate and a 9:1 mixture of acetonitrile/water was the solvent system used for further separation of Fraction VI. Four peaks were visualized using a UV-Vis detector. Based on the increased absorbance at 220 nm, 230 rim, and 280 rim, four fractions were collected.

Peak 3 was further purified by HPLC using an isocratic solvent system. A representative tracing from HPLC of repetitive injections of Peak 3 recorded at wavelengths of 220 mm, 230 nm, and 280 nm. Both peaks were collected and labeled as 3A and 3B respectively.

Peak 4 was further purified by HPLC using a gradient system. It was detected by increased UV absorbance readings at 220 nm, 230 nm, and 280 nm. Peak 4 was separated into two peaks and collected as Fractions 4A and 4B.

Submission of HPLC Fractions for Analysis by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS)

Fractions labeled as 3A and 3B were submitted to NMR and MS using chemical ionization and electron ionization. The molecular weight of Fraction 3A is estimated to be 279. Interpretation of the NMR spectra suggests a phenolic compound.

Fraction 3B has a molecular weight of 209 with an empirical formula consisting of C₁₀H₁₁NO₄. The substance para-hydroxyphenylacetylglycine has a similar molecular weight of 209. However, NMR data do not support the theory that parahydroxyphenylacetylglycine exists in the MNC complex. An ester structure found by NMR in the MNC complex is not found in the structure of para-hydroxyphenylacetylglycine. Also, para-hydroxyphenylacetylglycine has been only detected in Fraction VI.

Data from NMR support the conclusion that Peak 4 contains both an indole structure and a phenol structure.

SUMMARY

-   1. MNC from Fraction VI has been further purified using gradient and     isocratic HPLC into compounds 1,2, 3A, 3B, 4A, and 4B. -   2. The molecular weight of compound 3B is known at 209 (C₁₀0H₁₁NO₄). -   3. One structure with a molecular weight of 209 has been found in     Fraction VI. It has been identified as     para-hydroxyphenylacetylglycine. -   4. However, a unique compound with a phenylester structure and     having an empirical formula of C₁₀0H₁₁NO₄ best corresponds to the     data accumulated from NMR. -   5. Thus, a unique substance (which is part of the MNC complex     associated only with the denning phenomenon) is found in     Fraction VI. This unique substance also contains significant     biopotential for stimulation of osteoblasts.     Anticipated Treatment Results

Based upon studies with guinea pigs, bone cultures, black bears, and polar bears, the anticipated results of BDI treatment in humans follow.

Osteoporosis

Successful treatment of females or males suffering from osteoporosis or prevention of bone loss in them or in astronauts will be due to stimulation of osteoblasts (the cells that form bone), inhibition of resorption activity of osteoclasts, or simultaneous effects of osteoblasts and osteoclasts.

Thus, BDI becomes a potent, naturally occurring component to not only prevent osteoporosis but to increase size and strength of bone and successfully treat the debilitating condition of osteoporosis.

These changes may be evaluated by a general medical examination and optional diagnostic evaluations including radiographic assessment, measurement of the density of vertebral and other bones, prevention of bone fractures, and special assessment of skeletal remodeling activity.

Kidney Disease

Patients with chronic kidney disease or end stage renal failure may be treated so that the recycling of excess urea back into protein would result in the symptoms of kidney failure being reduced or abolished, to the extent that dialysis or kidney transplantation would not be needed.

Burns and Trauma

The prevention of excessive loss of protein from non-involved muscle and other tissues would treat patients with severe burns and trauma.

Muscle Atrophy

This treatment may maintain muscle mass in humans as they age and may prevent loss of muscle tissue in astronauts.

Obesity and Other Eating Disorders

The interfacing of increasing deposition of healthy lean tissue while eating less would have a pronounced favorable effect on the treatment of obesity in human beings. When the effective dose of BDI is adjusted for safety and to a degree that it promotes less food intake to a point of complete absence while preserving lean tissues, treatment of one of the most resistant disorders of human beings may be accomplished.

An anticipated treatment result, based on studies of hyperphagic black bears, would be to stimulate food intake in humans suffering from poor food intake such as anorexia nervosa.

General Health

In humans, the overall effects of BDI are expected to enhance general health while substantially reducing cost of health care.

Predictability and Correlatability of Comparable Results in Humans

While in vivo tests have not been made with regard to bone remodeling by the bear derived isolate of claim 1, in vitro tests have been done. Such in vitro tests are set forth in a recent April 1994 draft publication by the FDA. The publication is entitled “Guidelines for Pre-Clinical and Clinical Evaluation of Agents Used in the Prevention or Treatment of Post Menopausal Osteoporosis”. The draft was prepared by The Division of Metabolism and Endocrine Drug Products of the FDA, as indicated in April of 1994. The following shows a comparison between the guidelines (Page 4, Section IV) and results achieved with BDI.

Suggested FDA Guidelines BDI Test Results 1. At least one biochemical marker of 1. BDI isolated from summer fasting urine bone resorption. inhibits the production of tartrate resistant acid phosphatase in mouse calvaria organ cultures. Tartrate resistant acid phosphatase is produced by osteoclasts and serves as a sign of bone resorption (Lau, et al., 1987; Delmas, 1988). 2. At least one biochemical marker for 2. When added to an organ (bone) culture of bone formation. mouse calvaria, BD1 isolated from winter denning urine or from summer fasting urine produced a statistically significant production of alkaline phosphatase which represents stimulation of osteoblasts (Aurback, Marx, et. al., 1992; Delmas, 1988, 1993; Mundy, Roodman, 1991; Parviainen, Pirskanen, 1991; Stein, Lian, 1990, 1993; Quarles, Yokay, et al., 1992). 3. That alkaline phosphatase is the 3. When BDI was broken down into ten suggested biochemical marker for bone individual fractions, fractions V. VI, formation. and VII proved to be the most potent in stimulating statistically significant production of alkaline phosphatase by osteoblasts located in the bone of mouse calvaria. 4. A suggested biochemical marker of 4. Rather than using an indirect method to bone resorption is urinary pyridinium assess bone resorption, our studies have crosslinks. shown that BDI inhibits resorption in two ways - the conversions of bone marrow monocytes into osteoclasts, and by the inhibition of osteoclasts already functioning in bone resorptive cavities. 5. Measurement of serum osteocalcin (a specific marker of bone formation) is encouraged.

The foregoing results confirm in vitro bone remodeling consistent with the FDA outlined guidelines. Ongoing in vivo studies have confirmed the following.

Pre-Clinical in vivo Studies 1. Study conducted in an in vivo model 1. When compared with the untreated, such as the ovariectomized, osteoporotic ovariectomized rat, osteoporotic rat. ovariectomized rats that had been treated with DBI showed a 16-fold increase in bone mineral density of the femoral bone and a 4-fold increase in the vertebral bones when compared with bone mineral density of humans receiving therapeutic estrogen therapy over the same or trial period. 2. Histomorphometry or measurement of 2. Histomorphometry of the femoral and serum osteocalcium (a specific marker vertebral bones from the DBI treated, of bone formation) is encouraged. ovariectomized, osteoporatic rats is now underway.

The foregoing in vivo studies correlate with the FDA guidelines.

In addition, the subject matter of claim 1 has the ability to modulate the urea to creatinine ratio in urine of the guinea pig to values of 10 or less. Thus, tests were affirmative, and indicative of an increased ability of the guinea pig to recycle urea (Table 16). Bone mineral density in ovariectomized rats increased when those rats were treated with the subject matter of claim 1.

Nelson, Jones, et al. (1975) showed that urea is continually produced in the denning bear. Since the bear doesn't urinate, urea levels in blood, if unchecked, would result in high levels of urea (uremia) and death. Ahlquist, Nelson, et al. (1984) and Wolfe, Nelson et al. (1982, 1982a) showed that uremia is prevented by recycling the newly formed urea almost immediately back into protein from which it came. Nitrogen from urea was split off and attached to glycerol released from stored fat in adipose tissue. The newly formed amino acids were then incorporated in proteins such as albumin and fibrinogen.

Nelson, Beck, et al. (1984) showed that the rapid recycling of urea resulted in a decline of the level of urea in blood. When expressed as a ratio of urea to creatinine, the ratio decreased from 20 or more to less than 10. Such ratios were only found in denning bears who were not drinking or urinating. In catheterized urine specimens of denning bears, Nelson, Wahner, et al. (1973) showed when urea recycling was in process, the urea to creatinine ratio in urine was also reduced to values less than 10.

When BDI was injected into guinea pigs, urine U/C was decreased to values less than 10 indicative of similar urea recycling in guinea pigs as shown by denning bears. A strong indicator of suitability of bear originated materials for pharmacologic use in humans is the use of the bile salt produced by the bear, ursodeoxycholic acid (UDCA).

-   1. UDCA is safe and effective therapy for patients with cholesterol     gall stones (Rubin, Kowalski, et al., 1994). -   2. UDCA currently offers the best combination of efficacy and lack     of side effects in treatment of primary biliary cirrhosis and     reduces the need for liver transplants (Lim, Northfield 1994;     Poupon, Poupon, et al., 1994). -   3. UDCA improves liver function in primary sclerosing cholangitis of     the liver (Jazrawi, De Coestecker, et al., 1994). -   4. UDCA is a safe, well-tolerated, and efficacious treatment of     refractory chronic graft versus host disease of the liver occurring     in patients receiving bone marrow transplants (Fried, Murakawi, et     al., 1992). -   5. UDCA is a bear derivative acceptable and approved to be     administered to humans.

Accordingly, it is extrapolated that if one bear derivative is administered pharmaceutically to humans as a pharmacological product, another bear derivative will be similarly acceptable. This acceptability is reinforced by the cited tests with guinea pigs.

In summary, the conclusion reached after many years of study, observation of the phenomenon of bears, and predicated upon numerous publications set forth in the bibliography filed with this application, the predictability and correlatability to comparable results when administered to humans is present within the confines of the current disclosure.

Other Investigations

In addition to those described, investigations relating the close proximity of the BDI isolate with other normally appearing metabolic substances suggests that they are required to achieve action. Thus, BDI, the bear derived isolate alone, may require other metabolites to exert its action. Further portions of the entirety of the isolate may be combined or absorbed into these substances to exert action. This equivalency may be a function of these interactions and substantially produce the same result.

Summary of Present Discovery and Areas for Further Research

Already achieved as set forth above is the discovery of how the bear forms bone, even though existing in a state similar to post-menopausal women. The discovery reveals that BDI inhibits bone resorption by inhibiting the maturation of osteoclasts from bone marrow monocytes and by directly inhibiting functioning osteoclasts. The discovery has confirmed that a unique feature of BDI is that rather than inhibiting osteoblasts as current drugs do (and thus reducing bone production), BDI independently stimulates osteoblasts to form bone. Even though the bear inhibits osteoclasts, at the same time it independently stimulates osteoblasts to form bone. This novel, unique approach of direct osteoblast stimulation by BDI has been shown in cell and organ bone cultures. When current drugs on the market inhibit bone resorption by osteoclasts, osteoblast numbers and activity are also inhibited. BDI's unique ability to directly stimulate osteoblastic proliferation is demonstrated. Moreover, BDI directly stimulates fibroblastic activity which involves the matrix formation and production of bone stimulating factors. Again, no drugs on the market have this action. Finally, BDI stimulates bone formation in the ovariectomized rat, a model similar to post-menopausal women. GC/MS has established the identifiable ingredients present in BDI. Using countercurrent chromatography (CCC), fractions were developed that separated BDI into semi-purified fractional components that affect osteoblasts, osteoclasts, and fibroblasts. These discoveries include the potent Fractions V, VI, and VII that stimulate osteoblast and fibroblast proliferation and bone formation by osteoblasts. This is to the exclusion of the inhibition of osteoblastic activity of BDI found in Fraction III. Moreover, the discoveries of the constituents of Fractions V, VI, and VII by first producing them by CCC and then by determining their composition and concentration by GC/MS has led to further investigations. This includes the fact that bone resorption inhibiting activity of BDI is found mainly in the first three fractions of BDI as produced by CCC. Also, Fraction III inhibits osteoblasts directly.

Additionally, the potency of Fractions V, VI, and VII on forming bone in the osteoporotic rat can be calculated from the in vivo rat studies, the in vitro organ cultures of mouse calvarial bone and the cell cultures of osteoblasts.

Future Investigations

What is thus required is the following:

The combined potency of Fractions V, VI, and VII of BDI needs to be determined. This may result in the discovery of a unique substance that orchestrates all of the bone forming activity of BDI or in the fact that BDI represents a novel and unique combination of previously known as well as recently discovered new compounds. This substance or combination will be tested using in vitro and in vivo methods. This novel and unique substance or combination of substances will be synthesized and tested for bone forming activity in a model of the post-menopausal human, ovariectomized rats.

Other Bear Species

the effects of BDI as related to urea recycling extend from the black bear to include grizzly and polar bears. Both of these species demonstrate urea recycling as shown by a low blood urea to creatinine ratio when not drinking water or eating snow. No other mammal has this ability. If not drinking water, or if water is withheld, all other animals show an increase in blood urea and dehydration. Their urea to creatinine ratio rises above 20 and death will occur if water is not taken. Because of the effective urea recycling process, when not drinking or eating, black, grizzly, and polar bears protect their lean body mass, behave normally, and can be physically active. Since BDI induces denning phenomenon in guinea pigs (including urea recycling), BDI can be predicted to be similar in effects if obtained from urine or blood from grizzly or polar bears.

SCOPE OF THE INVENTION

It will be understood that within the scope of the invention as expressed in the appended claims, various changes in the details and materials which have been herein described and illustrated in order to explain the nature of the invention, may be made by those skilled in the art within the principle and scope of the invention as expressed in the appended claims. 

1. A method of treating obesity by administering to obese subjects a therapeutically effective amount of polypeptides having between about 12 and 13 amino acid residues in length and wherein the polypeptide comprises a compound selected from the group consisting of RTGEGFLVTGGGV (SEQ ID NO: 1) and VSKEGFLVTGGR (SEQ ID NO: 2).
 2. The method of claim 1 wherein the polypeptide comprises a compound comprising the following formula: RTGEGFLVTGGGV (SEQ ID NO: 1).
 3. The method of claim 1 wherein the polypeptide comprises a compound comprising the following formula: VSKEGFLVTGGR (SEQ ID NO: 2).
 4. The method of claim 1 wherein the polypeptide comprises a mixture of RTGEGFLVTGGGV (SEQ ID NO: 1) and VSKEGFLVTGGR (SEQ ID NO: 2).
 5. A composition for treating obesity comprising a therapeutically effective level of a polypeptide having between about 12 and 13 amino acid residues in length and wherein said polypeptide is selected from the group consisting of RTGEGFLVTGGGV (SEQ ID NO: 1) and VSKEGFLVTGGR (SEQ ID NO: 2) and a pharmaceutically acceptable carrier.
 6. The composition of claim 5 wherein the polypeptide comprises a compound comprising the following formula: RTGEGFLVTGGGV (SEQ ID NO: 1).
 7. The composition of claim 5 wherein the polypeptide comprises a compound comprising the following formula: VSKEGFLVTGGR (SEQ ID NO: 2).
 8. The composition of claim 5 wherein the polypeptide comprises a mixture of RTGEGFLVTGGGV (SEQ ID NO: 1) and VSKEGFLVTGGR (SEQ ID NO: 2). 